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# Ferroptosis in immunostimulation and immunosuppression

Daolin Tang, Guido Kroemer, Rui Kang

<figure><img src="image_2.png" /></figure>

## To cite this version:

Daolin Tang, Guido Kroemer, Rui Kang. Ferroptosis in immunostimulation and immunosuppression. Immunological Reviews, 2023, 321 (1), pp.199-210. 10.1111/imr.13235. hal-04599247

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# Ferroptosis in immunostimulation and immunosuppression

Daolin Tang, Guido Kroemer, Rui Kang

► To cite this version:

Daolin Tang, Guido Kroemer, Rui Kang. Ferroptosis in immunostimulation and immunosuppression. Immunological Reviews, 2023, 321 (1), pp.199-210. 10.1111/imr.13235. hal-04599247

HAL Id: hal-04599247

https://hal.science/hal-04599247v1

Submitted on 3 Jun 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
INVITED REVIEW

Immunological Reviews

<figure><img src="image_3.png" /></figure>

# Ferroptosis in immunostimulation and immunosuppression

Daolin Tang<fnref n="1" /> ID | Guido Kroemer<fnref n="2" />,<fnref n="3" />,<fnref n="4" /> ID | Rui Kang<fnref n="1" /> ID

**Correspondence**

Daolin Tang and Rui Kang, Department of Surgery, UT Southwestern Medical Center, Dallas, Texas 75390, USA.
Email: daolin.tang@utsouthwestern.edu and rui.kang@utsouthwestern.edu

Guido Kroemer, Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Université Paris Cité, Sorbonne Université, INSERM U1138, Institut Universitaire de France, Paris, France.
Email: kroemer@orange.fr

**Funding information**

National Institutes of Health, Grant/Award Number: R01CA211070, R01CA229275 and R01CA160417; Ligue contre le Cancer; Agence National de la Recherche (ANR)—Projets blancs; AMMICa, Grant/Award Number: US23/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); Equipex Onco-Pheno-Screen; European Joint Programme on Rare Diseases (EJPRD); European Research Council Advanced Investigator Award, Grant/Award Number: 101052444; European Union Horizon 2020 Projects Oncobiome, Prevalung, Grant/Award Number: 101095604; Crimson; Fondation Carrefour; Institut National du Cancer (INCa); Institut Universitaire de France; LabEx Immuno-Oncology, Grant/Award Number: ANR-18-IDEX-0001; Cancer Research ASPIRE Award from the Mark Foundation; RHU Immunolife; Seerave Foundation; SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); SIRIC Cancer Research and Personalized Medicine (CARPEM); IdEx Université de Paris, Grant/Award Number: ANR-18-IDEX-0001

**Summary**

Ferroptosis is a form of iron-dependent regulated cell death characterized by the accumulation of toxic lipid peroxides, particularly in the plasma membrane, leading to lytic cell death. While it plays a crucial role in maintaining the overall health and proper functioning of multicellular organisms, it can also contribute to tissue damage and pathological conditions. Although ferroptotic damage is generally recognized as an immunostimulatory process associated with the release of damage-associated molecular patterns (DAMPs), the occurrence of ferroptosis in immune cells or the release of immunosuppressive molecules can result in immune tolerance. Consequently, there is ongoing exploration of targeting the upstream signals or the machinery of ferroptosis to therapeutically enhance or inhibit the immune response. In addition to introducing the core molecular mechanisms of ferroptosis, we will focus on the immune characteristics of ferroptosis in pathological conditions, particularly in the context of infection, sterile inflammation, and tumor immunity.

**KEYWORDS**

ferroptosis, immunostimulation, immunosuppression, infection, sterile inflammation, tumor immunity

This article is part of a series of reviews covering Mechanisms of programmed cell death appearing in Volume 321 of *Immunological Reviews*.

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2023 The Authors. *Immunological Reviews* published by John Wiley & Sons Ltd.

<footnote n="1">¹Department of Surgery, UT Southwestern Medical Center, Dallas, Texas, USA</footnote>

<footnote n="2">²Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Université Paris Cité, Sorbonne Université, INSERM U1138, Institut Universitaire de France, Paris, France</footnote>

<footnote n="3">³Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France</footnote>

<footnote n="4">⁴Institut du Cancer Paris CARPEM, Department of Biology, Hôpital Européen Georges Pompidou, AP-HP, Paris, France</footnote>
<figure><img src="image_2.png" /></figure>

INVITED REVIEW

Immunological Reviews

<figure><img src="image_3.png" /></figure>

# Ferroptosis in immunostimulation and immunosuppression

Daolin Tang<fnref n="1" /> ID | Guido Kroemer<fnref n="2" />,<fnref n="3" />,<fnref n="4" /> ID | Rui Kang<fnref n="1" /> ID

**Correspondence**

Daolin Tang and Rui Kang, Department of Surgery, UT Southwestern Medical Center, Dallas, Texas 75390, USA.
Email: daolin.tang@utsouthwestern.edu and rui.kang@utsouthwestern.edu

Guido Kroemer, Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Université Paris Cité, Sorbonne Université, INSERM U1138, Institut Universitaire de France, Paris, France.
Email: kroemer@orange.fr

**Funding information**

National Institutes of Health, Grant/Award Number: R01CA211070, R01CA229275 and R01CA160417; Ligue contre le Cancer; Agence National de la Recherche (ANR)—Projets blancs; AMMICa, Grant/Award Number: US23/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); Equipex Onco-Pheno-Screen; European Joint Programme on Rare Diseases (EJPRD); European Research Council Advanced Investigator Award, Grant/Award Number: 101052444; European Union Horizon 2020 Projects Oncobiome, Prevalung, Grant/Award Number: 101095604; Crimson; Fondation Carrefour; Institut National du Cancer (INCa); Institut Universitaire de France; LabEx Immuno-Oncology, Grant/Award Number: ANR-18-IDEX-0001; Cancer Research ASPIRE Award from the Mark Foundation; RHU Immunolife; Seerave Foundation; SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); SIRIC Cancer Research and Personalized Medicine (CARPEM); IdEx Université de Paris, Grant/Award Number: ANR-18-IDEX-0001

**Summary**

Ferroptosis is a form of iron-dependent regulated cell death characterized by the accumulation of toxic lipid peroxides, particularly in the plasma membrane, leading to lytic cell death. While it plays a crucial role in maintaining the overall health and proper functioning of multicellular organisms, it can also contribute to tissue damage and pathological conditions. Although ferroptotic damage is generally recognized as an immunostimulatory process associated with the release of damage-associated molecular patterns (DAMPs), the occurrence of ferroptosis in immune cells or the release of immunosuppressive molecules can result in immune tolerance. Consequently, there is ongoing exploration of targeting the upstream signals or the machinery of ferroptosis to therapeutically enhance or inhibit the immune response. In addition to introducing the core molecular mechanisms of ferroptosis, we will focus on the immune characteristics of ferroptosis in pathological conditions, particularly in the context of infection, sterile inflammation, and tumor immunity.

**KEYWORDS**

ferroptosis, immunostimulation, immunosuppression, infection, sterile inflammation, tumor immunity

This article is part of a series of reviews covering Mechanisms of programmed cell death appearing in Volume 321 of *Immunological Reviews*.

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2023 The Authors. *Immunological Reviews* published by John Wiley & Sons Ltd.

<footnote n="1">¹Department of Surgery, UT Southwestern Medical Center, Dallas, Texas, USA</footnote>

<footnote n="2">²Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Université Paris Cité, Sorbonne Université, INSERM U1138, Institut Universitaire de France, Paris, France</footnote>

<footnote n="3">³Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France</footnote>

<footnote n="4">⁴Institut du Cancer Paris CARPEM, Department of Biology, Hôpital Européen Georges Pompidou, AP-HP, Paris, France</footnote>
# 1 INTRODUCTION

Cell death is a natural and essential part of the cellular life cycle, playing a fundamental role in the development, maintenance, and overall health of multicellular organisms. Cells can die through different molecular mechanisms that are classified into accidental cell death (ACD) and regulated cell death (RCD).¹ Unlike ACD, RCD is a controlled process with multiple subroutines that are time-dependent and ultimately halt cell functions.² In 1972, pathologists observed a specific type of cell death in human or rat tissues characterized by condensed and fragmented cells and nuclei.³ They named this cell death process “apoptosis.” As research in cell death progresses, the focus has expanded from the very well-characterized process of apoptosis to include an increasing number of non-apoptotic cell death modalities.

Among these modalities, iron-dependent and non-apoptotic ferroptosis have garnered significant attention in translational medicine.⁴ The concept of ferroptosis was coined in 2012 during the screening of small molecules that selectively induced cell death in cancer cell lines (HT1080 and Calu1) harboring oncogenic RAS mutations.⁵ We now know that ferroptosis can also occur in normal cells or tissues, independent of mutant RAS activation, and its induction is involved in the disruption of oxidative and antioxidant balance.⁶ It is noteworthy that the core steps of ferroptosis, including glutathione depletion, glutathione peroxidase 4 (GPX4) inactivation, and lipid peroxidation, closely resemble those of oxytosis.⁷ Oxytosis, initially described in a neuronal cell line (N18-RE-105) during the late 1980s, represents a non-excitotoxic pathway for cell death induced by glutamate.⁸

In the immune system, cell death plays a pivotal role in the elimination of damaged, infected, or abnormal cells. RCD allows the removal of cells that pose a threat to the overall health and function of the organism. Proper regulation of cell death is crucial for maintaining immune tolerance and homeostasis.⁹ Dysregulation of RCD pathways, including ferroptosis, can lead to immune dysfunctions, such as excessive inflammation, autoimmune diseases, or immunodeficiency.¹⁰ Understanding the delicate balance between cell death and immune tolerance is vital for preserving immune homeostasis and preventing immune-related disorders.¹¹

In this narrative review, we aim to provide an overview of the basic principles of ferroptosis while delving into the dual role of ferroptosis in both immunostimulation and immunosuppression. By exploring the intricate pathways associated with ferroptosis,¹² we provide new insights into disease pathogenesis and identify potential therapeutic targets. The interplay between ferroptosis and immune responses adds another layer of complexity to our understanding of the immune system and its implication in various diseases.

# 2 MOLECULAR MACHINERY: KEY HALLMARKS OF FERROPTOSIS

Ferroptosis can occur through two major pathways: the extrinsic or transporter-mediated pathway that can be activated by the

inhibition solute carrier family 7 member 11 (SLC7A11), and the intrinsic or enzyme-related pathway that results from the inhibition of GPX4 (Figure 1).¹³ The core molecular machinery and signaling regulation have primarily been identified in cancer cells using small molecular compounds like erastin and RSL3, which inhibit SLC7A11 and GPX4, respectively.⁵,¹⁴ SLC7A11 is the main functional component of the amino acid transporter system xc⁻ located on the cell membrane. This transporter plays a crucial role in the transport of cystine (a sulfur-containing amino acid) and glutamate (an important neurotransmitter in the central nervous system). The transporter exchanges intracellular glutamate with extracellular cystine in a one-to-one stoichiometry. This exchange is vital for maintaining cellular redox balance and providing cysteine, a precursor for the synthesis of glutathione, an essential antioxidant that acts as a cofactor for GPX4.¹⁵ GPX4 stands out among glutathione peroxidases due to its distinctive ability to directly reduce and detoxify lipid hydroperoxides.¹⁶ These lipid hydroperoxides are generated through the oxidation of polyunsaturated fatty acids (PUFA), which are fatty acids containing two or more double bonds between carbon atoms, primarily found in cell membranes.¹⁷ This property renders GPX4 particularly important in protecting cells from oxidative stress-induced lipid peroxidation, which can lead to cell damage, inflammation, and various diseases.¹⁵

Unlike other classical cell death inducers that typically activate executors of cell death, the initiation of ferroptosis primarily occurs through blocking a cell death-inhibitory system involving antioxidant mechanisms. In addition to erastin and RSL3, several drugs (e.g., sorafenib and sulfasalazine) or yet-to-be developed compounds (e.g., ML210 and ML162) have been found to inhibit SLC7A11 or GPX4 activity, thus inducing lipid peroxidation and triggering ferroptosis (Figure 1). GPX4 is ubiquitously expressed in various tissues. It can be found in the cytosol, mitochondria, and nucleus.¹⁸ However, current evidence indicates that its primary role is to inhibit ferroptosis specifically within the cytosol.¹⁴ Other antioxidant enzymes, such as apoptosis-inducing factor mitochondria-associated 2 (AIFM2; also known as FSP1),¹⁹ dihydroorotate dehydrogenase (DHODH),²⁰ GTP cyclohydrolase 1 (GCH1),²¹ and microsomal glutathione S-transferase 1 (MGST1),²² play GPX4-dependent or -independent roles in inhibiting ferroptosis in cancer or non-cancer cells. These observations highlight the importance of understanding how different antioxidant systems collaborate to form an integrated defense against ferroptotic damage. Notably, NFE2 like BZIP transcription factor 2 (NFE2L2; also known as NRF2), a nuclear transcription factor activated in response to oxidative stress, plays a central role in transactivating anti-ferroptotic genes,²³ indicating the existence of a transcriptional machinery that shapes the response to ferroptosis.

Another distinguishing characteristic of ferroptosis, compared to other forms of cell death, is that the effector molecules of ferroptosis appear to be toxic lipids rather than proteins.²⁴,²⁵ In apoptosis, necroptosis, and pyroptosis, caspases, mixed-lineage kinase domain-like pseudokinase (MLKL), and gasdermin D (GSDMD), respectively, serve as lethal effector molecules due to their ability to cleave proteins or to form pores in the cell membrane.² However, these
# 1 INTRODUCTION

Cell death is a natural and essential part of the cellular life cycle, playing a fundamental role in the development, maintenance, and overall health of multicellular organisms. Cells can die through different molecular mechanisms that are classified into accidental cell death (ACD) and regulated cell death (RCD).¹ Unlike ACD, RCD is a controlled process with multiple subroutines that are time-dependent and ultimately halt cell functions.² In 1972, pathologists observed a specific type of cell death in human or rat tissues characterized by condensed and fragmented cells and nuclei.³ They named this cell death process “apoptosis.” As research in cell death progresses, the focus has expanded from the very well-characterized process of apoptosis to include an increasing number of non-apoptotic cell death modalities.

Among these modalities, iron-dependent and non-apoptotic ferroptosis have garnered significant attention in translational medicine.⁴ The concept of ferroptosis was coined in 2012 during the screening of small molecules that selectively induced cell death in cancer cell lines (HT1080 and Calu1) harboring oncogenic RAS mutations.⁵ We now know that ferroptosis can also occur in normal cells or tissues, independent of mutant RAS activation, and its induction is involved in the disruption of oxidative and antioxidant balance.⁶ It is noteworthy that the core steps of ferroptosis, including glutathione depletion, glutathione peroxidase 4 (GPX4) inactivation, and lipid peroxidation, closely resemble those of oxytosis.⁷ Oxytosis, initially described in a neuronal cell line (N18-RE-105) during the late 1980s, represents a non-excitotoxic pathway for cell death induced by glutamate.⁸

In the immune system, cell death plays a pivotal role in the elimination of damaged, infected, or abnormal cells. RCD allows the removal of cells that pose a threat to the overall health and function of the organism. Proper regulation of cell death is crucial for maintaining immune tolerance and homeostasis.⁹ Dysregulation of RCD pathways, including ferroptosis, can lead to immune dysfunctions, such as excessive inflammation, autoimmune diseases, or immunodeficiency.¹⁰ Understanding the delicate balance between cell death and immune tolerance is vital for preserving immune homeostasis and preventing immune-related disorders.¹¹

In this narrative review, we aim to provide an overview of the basic principles of ferroptosis while delving into the dual role of ferroptosis in both immunostimulation and immunosuppression. By exploring the intricate pathways associated with ferroptosis,¹² we provide new insights into disease pathogenesis and identify potential therapeutic targets. The interplay between ferroptosis and immune responses adds another layer of complexity to our understanding of the immune system and its implication in various diseases.

# 2 MOLECULAR MACHINERY: KEY HALLMARKS OF FERROPTOSIS

Ferroptosis can occur through two major pathways: the extrinsic or transporter-mediated pathway that can be activated by the

inhibition solute carrier family 7 member 11 (SLC7A11), and the intrinsic or enzyme-related pathway that results from the inhibition of GPX4 (Figure 1).¹³ The core molecular machinery and signaling regulation have primarily been identified in cancer cells using small molecular compounds like erastin and RSL3, which inhibit SLC7A11 and GPX4, respectively.⁵,¹⁴ SLC7A11 is the main functional component of the amino acid transporter system xc⁻ located on the cell membrane. This transporter plays a crucial role in the transport of cystine (a sulfur-containing amino acid) and glutamate (an important neurotransmitter in the central nervous system). The transporter exchanges intracellular glutamate with extracellular cystine in a one-to-one stoichiometry. This exchange is vital for maintaining cellular redox balance and providing cysteine, a precursor for the synthesis of glutathione, an essential antioxidant that acts as a cofactor for GPX4.¹⁵ GPX4 stands out among glutathione peroxidases due to its distinctive ability to directly reduce and detoxify lipid hydroperoxides.¹⁶ These lipid hydroperoxides are generated through the oxidation of polyunsaturated fatty acids (PUFA), which are fatty acids containing two or more double bonds between carbon atoms, primarily found in cell membranes.¹⁷ This property renders GPX4 particularly important in protecting cells from oxidative stress-induced lipid peroxidation, which can lead to cell damage, inflammation, and various diseases.¹⁵

Unlike other classical cell death inducers that typically activate executors of cell death, the initiation of ferroptosis primarily occurs through blocking a cell death-inhibitory system involving antioxidant mechanisms. In addition to erastin and RSL3, several drugs (e.g., sorafenib and sulfasalazine) or yet-to-be developed compounds (e.g., ML210 and ML162) have been found to inhibit SLC7A11 or GPX4 activity, thus inducing lipid peroxidation and triggering ferroptosis (Figure 1). GPX4 is ubiquitously expressed in various tissues. It can be found in the cytosol, mitochondria, and nucleus.¹⁸ However, current evidence indicates that its primary role is to inhibit ferroptosis specifically within the cytosol.¹⁴ Other antioxidant enzymes, such as apoptosis-inducing factor mitochondria-associated 2 (AIFM2; also known as FSP1),¹⁹ dihydroorotate dehydrogenase (DHODH),²⁰ GTP cyclohydrolase 1 (GCH1),²¹ and microsomal glutathione S-transferase 1 (MGST1),²² play GPX4-dependent or -independent roles in inhibiting ferroptosis in cancer or non-cancer cells. These observations highlight the importance of understanding how different antioxidant systems collaborate to form an integrated defense against ferroptotic damage. Notably, NFE2 like BZIP transcription factor 2 (NFE2L2; also known as NRF2), a nuclear transcription factor activated in response to oxidative stress, plays a central role in transactivating anti-ferroptotic genes,²³ indicating the existence of a transcriptional machinery that shapes the response to ferroptosis.

Another distinguishing characteristic of ferroptosis, compared to other forms of cell death, is that the effector molecules of ferroptosis appear to be toxic lipids rather than proteins.²⁴,²⁵ In apoptosis, necroptosis, and pyroptosis, caspases, mixed-lineage kinase domain-like pseudokinase (MLKL), and gasdermin D (GSDMD), respectively, serve as lethal effector molecules due to their ability to cleave proteins or to form pores in the cell membrane.² However, these
<figure><img src="image_4.png" /><figcaption>**FIGURE 1** Core molecular mechanism of ferroptosis. Ferroptosis is an oxidative cell death process driven by lipid peroxidation. Several key components contribute to this process. The classical ferroptosis inducers include inhibitors of the cell membrane system xc⁻ and intracellular glutathione peroxidase 4 (GPX4). The system xc⁻ acts as an amino acid antiporter composed of solute carrier family 7 member 11 (SLC7A11) and solute carrier family 3 member 2 (SLC3A2). This antiporter facilitates the uptake of cystine, an oxidized form of cysteine, which is crucial for the subsequent synthesis of glutathione (GSH) by the enzyme glutamate-cysteine ligase catalytic subunit (GCLC). GCLC serves as the first rate-limiting enzyme in the process of GSH synthesis. GPX4, an essential selenocysteine-containing enzyme, plays a crucial role in the reduction of lipid peroxides by utilizing reduced GSH. This enzymatic activity leads to the production of lipid alcohols, contributing to the protection of cells against oxidative stress. The transferrin receptor (TFRC) plays a significant role in mediating the entry of Fe³⁺ into the cell. Once inside, Fe³⁺ is reduced to Fe²⁺ and transported across the endosomal membrane via solute carrier family 11 member 2 (SLC11A2, also known as DMT1). The iron-mediated Fenton reaction generates reactive oxygen species (ROS), leading to the oxidation of polyunsaturated fatty acids (PUFAs), the major components of cell membranes. Additionally, enzymes involved in lipid peroxidation, such as lipoxygenase (ALOX) and cytochrome P450 oxidoreductase (POR), rely on iron for their activity. PUFA metabolism follows distinct pathways, including the acyl-CoA synthetase long chain family member 4 (ACSL4)-lysophosphatidylcholine acyltransferase 3 (LPCAT3) pathway and the ACSL4-sterol O-acyltransferase 1 (SOAT1) pathway, contributing to the generation of PUFA-phosphatidylethanolamines (PEs) and PUFA-cholesteryl esters (CES), respectively. The sensitivity of ferroptosis can be enhanced by selective autophagy processes like ferritinophagy and lipophagy, which lead to the accumulation of free iron and PUFAs. Finally, the endosomal sorting complexes required for transport (ESCRT)-III machinery, comprising various proteins like charged multivesicular body protein 5 (CHMP5) and charged multivesicular body protein 6 (CHMP6), participates in the repair of membrane damage caused by toxic lipids.</figcaption></figure>

traditional cell death mediators are not required for ferroptosis.⁵
Instead, the accumulation of lipid peroxidation products, such as 4-hydroxynonenal (4HNE) and malondialdehyde (MDA), can subvert cell membrane structure and function, ultimately leading to membrane rupture and cell death.²⁶,²⁷ Ferrostatin-1 and liproxstatin-1 are widely used ferroptosis inhibitors due to their ability to act as radical-trapping antioxidants, thereby slowing down the accumulation of lipid hydroperoxides.²⁸

The activation of lipid peroxidation in ferroptosis involves specific enzymes, such as those from the lipoxygenase (ALOX) family and cytochrome P450 oxidoreductase (POR). Interestingly, ALOX and POR play independent roles in mediating lipid peroxidation for ferroptosis.²⁹,³⁰ Although the threshold to distinguish physiological and pathological concentrations of 4HNE or MDA remains unclear, the loss of membrane repair machinery, such as endosomal sorting complexes required for transport (ESCRT)-III, can impair membrane

fluidity, accelerate cell edema, and ultimately result in ferroptotic cell death.³¹,³² Thus, genetic depletion of the core components of the membrane repair machinery, such as charged multivesicular body protein 5 (CHMP5) and charged multivesicular body protein 6 (CHMP6), can enhance ferroptosis-mediated tumor suppression in human cancer cells (PANC1 and HepG2).³¹

The sensitivity of cells to ferroptosis induction can be influenced at multiple levels, with various steps of iron or lipid metabolism controlling the levels of ferroptosis. Excessive iron not only produces reactive oxygen species (ROS) through the Fenton reaction but also activates iron-dependent lipid peroxidation enzymes, such as ALOXs or POR.³³ These processes collectively enhance the sensitivity to ferroptosis. Similarly, the accumulation of lipids, especially PUFAs primarily localized in cell membranes, provides targets for lipid peroxidation under oxidative stress. This process is driven by the acyl-CoA synthetase long chain family member 4 (ACSL4)-lysophosphatidylcholine acyltransferase 3
<figure><img src="image_4.png" /><figcaption>**FIGURE 1** Core molecular mechanism of ferroptosis. Ferroptosis is an oxidative cell death process driven by lipid peroxidation. Several key components contribute to this process. The classical ferroptosis inducers include inhibitors of the cell membrane system xc⁻ and intracellular glutathione peroxidase 4 (GPX4). The system xc⁻ acts as an amino acid antiporter composed of solute carrier family 7 member 11 (SLC7A11) and solute carrier family 3 member 2 (SLC3A2). This antiporter facilitates the uptake of cystine, an oxidized form of cysteine, which is crucial for the subsequent synthesis of glutathione (GSH) by the enzyme glutamate-cysteine ligase catalytic subunit (GCLC). GCLC serves as the first rate-limiting enzyme in the process of GSH synthesis. GPX4, an essential selenocysteine-containing enzyme, plays a crucial role in the reduction of lipid peroxides by utilizing reduced GSH. This enzymatic activity leads to the production of lipid alcohols, contributing to the protection of cells against oxidative stress. The transferrin receptor (TFRC) plays a significant role in mediating the entry of Fe³⁺ into the cell. Once inside, Fe³⁺ is reduced to Fe²⁺ and transported across the endosomal membrane via solute carrier family 11 member 2 (SLC11A2, also known as DMT1). The iron-mediated Fenton reaction generates reactive oxygen species (ROS), leading to the oxidation of polyunsaturated fatty acids (PUFAs), the major components of cell membranes. Additionally, enzymes involved in lipid peroxidation, such as lipoxygenase (ALOX) and cytochrome P450 oxidoreductase (POR), rely on iron for their activity. PUFA metabolism follows distinct pathways, including the acyl-CoA synthetase long chain family member 4 (ACSL4)-lysophosphatidylcholine acyltransferase 3 (LPCAT3) pathway and the ACSL4-sterol O-acyltransferase 1 (SOAT1) pathway, contributing to the generation of PUFA-phosphatidylethanolamines (PEs) and PUFA-cholesteryl esters (CES), respectively. The sensitivity of ferroptosis can be enhanced by selective autophagy processes like ferritinophagy and lipophagy, which lead to the accumulation of free iron and PUFAs. Finally, the endosomal sorting complexes required for transport (ESCRT)-III machinery, comprising various proteins like charged multivesicular body protein 5 (CHMP5) and charged multivesicular body protein 6 (CHMP6), participates in the repair of membrane damage caused by toxic lipids.</figcaption></figure>

traditional cell death mediators are not required for ferroptosis.⁵
Instead, the accumulation of lipid peroxidation products, such as 4-hydroxynonenal (4HNE) and malondialdehyde (MDA), can subvert cell membrane structure and function, ultimately leading to membrane rupture and cell death.²⁶,²⁷ Ferrostatin-1 and liproxstatin-1 are widely used ferroptosis inhibitors due to their ability to act as radical-trapping antioxidants, thereby slowing down the accumulation of lipid hydroperoxides.²⁸

The activation of lipid peroxidation in ferroptosis involves specific enzymes, such as those from the lipoxygenase (ALOX) family and cytochrome P450 oxidoreductase (POR). Interestingly, ALOX and POR play independent roles in mediating lipid peroxidation for ferroptosis.²⁹,³⁰ Although the threshold to distinguish physiological and pathological concentrations of 4HNE or MDA remains unclear, the loss of membrane repair machinery, such as endosomal sorting complexes required for transport (ESCRT)-III, can impair membrane

fluidity, accelerate cell edema, and ultimately result in ferroptotic cell death.³¹,³² Thus, genetic depletion of the core components of the membrane repair machinery, such as charged multivesicular body protein 5 (CHMP5) and charged multivesicular body protein 6 (CHMP6), can enhance ferroptosis-mediated tumor suppression in human cancer cells (PANC1 and HepG2).³¹

The sensitivity of cells to ferroptosis induction can be influenced at multiple levels, with various steps of iron or lipid metabolism controlling the levels of ferroptosis. Excessive iron not only produces reactive oxygen species (ROS) through the Fenton reaction but also activates iron-dependent lipid peroxidation enzymes, such as ALOXs or POR.³³ These processes collectively enhance the sensitivity to ferroptosis. Similarly, the accumulation of lipids, especially PUFAs primarily localized in cell membranes, provides targets for lipid peroxidation under oxidative stress. This process is driven by the acyl-CoA synthetase long chain family member 4 (ACSL4)-lysophosphatidylcholine acyltransferase 3
(LPCAT3) pathway, where free PUFAs are ligated with coenzyme A (CoA) to produce PUFA-CoAs for subsequent incorporation into phosphatidylethanolamines (PEs).³⁴,³⁵ In contrast, activation of the ACSL4-sterol O-acyltransferase 1 (SOAT1) pathway produces PUFA-cholesteryl esters (CEs) instead of PUFA-PEs, contributing to lipid peroxidation in soluble carrier family 47 member 1 (SLC47A1)-deficient human pancreatic cancer cells.³⁶ Thus, the lipid flippase SLC47A1 on the cell membrane may serve as a metabolic checkpoint influencing the induction of various ferroptosis pathways. It is worth noting that ACSL4-independent ferroptosis has been reported in certain cases, such as photodynamic therapy in human HeLa cancer cells.³⁷ In addition, ACSL3-dependent monounsaturated fatty acid (MUFA) production completely inhibits PUFA-dependent ferroptosis in HT1080 cells,³⁸ suggesting that different lipid species may have antagonistic effects.

In addition to direct enzymatic processes, iron or lipid metabolism can be regulated through degradation pathways. Specifically, the activation of selective autophagy, such as ferritinophagy³⁹ or lipophagy,⁴⁰ promotes the degradation of ferritin (an iron storage protein) in pancreatic cancer cells and mouse embryonic fibroblasts, as well as the catabolism of lipid droplets in liver cancer cells. Lipid droplets are cellular organelles that store lipids, primarily neutral lipids like triglycerides and cholesterol esters. During this degradation process, free iron and lipids are released, making them available for subsequent lipid peroxidation. These findings support the notion that ferroptosis is an autophagy-dependent cell death mechanism.⁴¹ However, autophagy can also inhibit ferroptosis in specific contexts, such as asthma.⁴² Gaining a deeper understanding of the heterogeneity and plasticity of the ferroptosis machinery is crucial for identifying pathways that may be targeted for disease modulation in specific circumstances.

## 3 | IMMUNE CONSEQUENCE: DUAL ROLE OF FERROPTOSIS

In many cases, ferroptosis exerts immunostimulatory effects, triggering immune responses and promoting inflammation (Figure 2). However, it is important to acknowledge that the occurrence of ferroptosis in immune cells can also lead to immunosuppression (Figure 3). The immunostimulatory or immunosuppression effects of ferroptosis may have both beneficial and detrimental consequences depending on context. In certain situations, ferroptosis can contribute to immune defense against pathogens or tumor cells, while in other instances, it may promote chronic inflammation, tissue damage, or autoimmune responses. In this section, our focus is to explore how ferroptotic stress influences sterile inflammation, infection by pathogens, and tumor immunosurveillance.

### 3.1 | Sterile inflammation

Sterile inflammation is triggered by endogenous molecules released from damaged or stressed cells rather than by invading pathogens.⁴³

This type of inflammation can arise from various factors, including tissue injury, trauma, autoimmune reactions, metabolic dysregulation, or exposure to certain environmental agents.

Studies using conditional Gpx4⁻/⁻ mice, which lack the antioxidant enzyme GPX4, have provided valuable insights into the involvement of ferroptosis in sterile inflammatory diseases. For instance, the specific loss of Gpx4 in the pancreas,⁴⁴ kidney,⁴⁵ liver,⁴⁶ or intestine⁴⁷ from mice has been demonstrated to induce or increase susceptibility to experimental pancreatitis, acute renal failure, liver necrosis, and Crohn's disease, respectively. Pancreatitis is an inflammatory disease characterized by the premature activation of digestive enzymes, such as trypsin. Loss of Gpx4 leads to increased sensitivity of pancreatic acinar cells to ferroptosis due to proteasome 26S subunit ubiquitin receptor, non-ATPase 4 (PSMD4)-dependent GPX4 protein degradation.⁴⁴ Oxi-lipidomics analysis of Gpx4⁻/⁻ kidneys reveals complex lipid oxidation patterns in phosphatidylcholine, PE, as well as in cardiolipin, a mitochondria-specific phospholipid.⁴⁵ In Gpx4⁻/⁻ livers, there is an upregulation of NFE2L2-targeted antioxidant genes as a defense mechanism.⁴⁶ Additionally, Gpx4-deficient small intestinal epithelial cells exhibit an inflammatory response upon exposure to PUFAs, resulting in the production of interleukin 6 (IL6) and C-X-C motif chemokine ligand 1 (CXCL1), thereby mediating Crohn's disease.⁴⁷ Furthermore, mice with Gpx4 haploinsufficiency in neutrophils develop spontaneous lupus-like disease characterized by the presence of autoantibodies, neutropenia, skin lesions, and proteinuria.⁴⁸ These pathological processes observed in Gpx4-deficient mice can be successfully inhibited by ferroptosis inhibitors, such as ferrostatin-1, liproxstatin-1, or vitamin E.

Additionally, several studies have demonstrated that ferrostatin-1 or liproxstatin-1 can protect against tissue ischemia-reperfusion injury, which occurs when blood supply to an organ or tissue is initially restricted and subsequently restored.⁴⁹⁻⁵³ Pharmacological inhibition of ferroptosis confers protection against experimental diabetes⁵⁴ and non-alcoholic fatty liver disease,⁵⁵ the most common manifestation of metabolic liver disease. These findings further underscore the broad role of ferroptotic damage in sterile inflammatory diseases.

Mechanistically, ferroptosis can induce an inflammatory response through the release of intracellular contents, including damage-associated molecular patterns (DAMPs) (Figure 2).⁴³ The release of DAMPs during ferroptosis has the potential to trigger immune activation and inflammation. These DAMPs usually activate pattern recognition receptors (PRRs) on immune cells, leading to the production of pro-inflammatory cytokines and recruitment of immune cells. For example, one well-known DAMP released during ferroptosis is high mobility group box 1 (HMGB1), which can activate immune cells and promote inflammation.⁵⁶ Moreover, another specific DAMP released during ferroptosis is decorin (DCN), which is recognized as a unique marker of ferroptotic death.⁵⁷ Both extracellular HMGB1 and DCN are agonists of advanced glycosylation end-product specific receptor (AGER; also known as RAGE) in macrophages.⁵⁶,⁵⁷ AGER activation subsequently triggers the production and release of pro-inflammatory cytokines, including IL6 and
(LPCAT3) pathway, where free PUFAs are ligated with coenzyme A (CoA) to produce PUFA-CoAs for subsequent incorporation into phosphatidylethanolamines (PEs).³⁴,³⁵ In contrast, activation of the ACSL4-sterol O-acyltransferase 1 (SOAT1) pathway produces PUFA-cholesteryl esters (CEs) instead of PUFA-PEs, contributing to lipid peroxidation in soluble carrier family 47 member 1 (SLC47A1)-deficient human pancreatic cancer cells.³⁶ Thus, the lipid flippase SLC47A1 on the cell membrane may serve as a metabolic checkpoint influencing the induction of various ferroptosis pathways. It is worth noting that ACSL4-independent ferroptosis has been reported in certain cases, such as photodynamic therapy in human HeLa cancer cells.³⁷ In addition, ACSL3-dependent monounsaturated fatty acid (MUFA) production completely inhibits PUFA-dependent ferroptosis in HT1080 cells,³⁸ suggesting that different lipid species may have antagonistic effects.

In addition to direct enzymatic processes, iron or lipid metabolism can be regulated through degradation pathways. Specifically, the activation of selective autophagy, such as ferritinophagy³⁹ or lipophagy,⁴⁰ promotes the degradation of ferritin (an iron storage protein) in pancreatic cancer cells and mouse embryonic fibroblasts, as well as the catabolism of lipid droplets in liver cancer cells. Lipid droplets are cellular organelles that store lipids, primarily neutral lipids like triglycerides and cholesterol esters. During this degradation process, free iron and lipids are released, making them available for subsequent lipid peroxidation. These findings support the notion that ferroptosis is an autophagy-dependent cell death mechanism.⁴¹ However, autophagy can also inhibit ferroptosis in specific contexts, such as asthma.⁴² Gaining a deeper understanding of the heterogeneity and plasticity of the ferroptosis machinery is crucial for identifying pathways that may be targeted for disease modulation in specific circumstances.

## 3 | IMMUNE CONSEQUENCE: DUAL ROLE OF FERROPTOSIS

In many cases, ferroptosis exerts immunostimulatory effects, triggering immune responses and promoting inflammation (Figure 2). However, it is important to acknowledge that the occurrence of ferroptosis in immune cells can also lead to immunosuppression (Figure 3). The immunostimulatory or immunosuppression effects of ferroptosis may have both beneficial and detrimental consequences depending on context. In certain situations, ferroptosis can contribute to immune defense against pathogens or tumor cells, while in other instances, it may promote chronic inflammation, tissue damage, or autoimmune responses. In this section, our focus is to explore how ferroptotic stress influences sterile inflammation, infection by pathogens, and tumor immunosurveillance.

### 3.1 | Sterile inflammation

Sterile inflammation is triggered by endogenous molecules released from damaged or stressed cells rather than by invading pathogens.⁴³

This type of inflammation can arise from various factors, including tissue injury, trauma, autoimmune reactions, metabolic dysregulation, or exposure to certain environmental agents.

Studies using conditional Gpx4⁻/⁻ mice, which lack the antioxidant enzyme GPX4, have provided valuable insights into the involvement of ferroptosis in sterile inflammatory diseases. For instance, the specific loss of Gpx4 in the pancreas,⁴⁴ kidney,⁴⁵ liver,⁴⁶ or intestine⁴⁷ from mice has been demonstrated to induce or increase susceptibility to experimental pancreatitis, acute renal failure, liver necrosis, and Crohn's disease, respectively. Pancreatitis is an inflammatory disease characterized by the premature activation of digestive enzymes, such as trypsin. Loss of Gpx4 leads to increased sensitivity of pancreatic acinar cells to ferroptosis due to proteasome 26S subunit ubiquitin receptor, non-ATPase 4 (PSMD4)-dependent GPX4 protein degradation.⁴⁴ Oxi-lipidomics analysis of Gpx4⁻/⁻ kidneys reveals complex lipid oxidation patterns in phosphatidylcholine, PE, as well as in cardiolipin, a mitochondria-specific phospholipid.⁴⁵ In Gpx4⁻/⁻ livers, there is an upregulation of NFE2L2-targeted antioxidant genes as a defense mechanism.⁴⁶ Additionally, Gpx4-deficient small intestinal epithelial cells exhibit an inflammatory response upon exposure to PUFAs, resulting in the production of interleukin 6 (IL6) and C-X-C motif chemokine ligand 1 (CXCL1), thereby mediating Crohn's disease.⁴⁷ Furthermore, mice with Gpx4 haploinsufficiency in neutrophils develop spontaneous lupus-like disease characterized by the presence of autoantibodies, neutropenia, skin lesions, and proteinuria.⁴⁸ These pathological processes observed in Gpx4-deficient mice can be successfully inhibited by ferroptosis inhibitors, such as ferrostatin-1, liproxstatin-1, or vitamin E.

Additionally, several studies have demonstrated that ferrostatin-1 or liproxstatin-1 can protect against tissue ischemia-reperfusion injury, which occurs when blood supply to an organ or tissue is initially restricted and subsequently restored.⁴⁹⁻⁵³ Pharmacological inhibition of ferroptosis confers protection against experimental diabetes⁵⁴ and non-alcoholic fatty liver disease,⁵⁵ the most common manifestation of metabolic liver disease. These findings further underscore the broad role of ferroptotic damage in sterile inflammatory diseases.

Mechanistically, ferroptosis can induce an inflammatory response through the release of intracellular contents, including damage-associated molecular patterns (DAMPs) (Figure 2).⁴³ The release of DAMPs during ferroptosis has the potential to trigger immune activation and inflammation. These DAMPs usually activate pattern recognition receptors (PRRs) on immune cells, leading to the production of pro-inflammatory cytokines and recruitment of immune cells. For example, one well-known DAMP released during ferroptosis is high mobility group box 1 (HMGB1), which can activate immune cells and promote inflammation.⁵⁶ Moreover, another specific DAMP released during ferroptosis is decorin (DCN), which is recognized as a unique marker of ferroptotic death.⁵⁷ Both extracellular HMGB1 and DCN are agonists of advanced glycosylation end-product specific receptor (AGER; also known as RAGE) in macrophages.⁵⁶,⁵⁷ AGER activation subsequently triggers the production and release of pro-inflammatory cytokines, including IL6 and
<figure><img src="image_5.png" /><figcaption>FIGURE 2 Mechanism of ferroptosis-mediated sterile inflammation. Ferroptotic cells can release various damage-associated molecular patterns (DAMPs) that trigger an inflammatory response. For instance, high mobility group box 1 (HMGB1) and decorin (DCN) can bind to advanced glycosylation end-product specific receptor (AGER), activating the nuclear factor kappa B (NF-κB) pathway. This process involves the adaptor protein diaphanous related formin 1 (DIAPH1) and signal transduction through the mitogen-activated protein kinase (MAPK) pathway. Additionally, excessive lipid peroxidation leads to the release of 4-hydroxynonenal (4HNE) and oxidized phospholipids (OxPL). These molecules can activate toll like receptor 4 (TLR4) signaling, resulting in the induction of proinflammatory cytokines (such as interleukin 6 [IL6] and tumor necrosis factor [TNF]) and type I interferon (IFN, such as IFNA and IFNB) through different adaptor proteins and signaling pathways. In general, MYD88 innate immune signal transduction adaptor (MYD88) is required for IKK-dependent NF-κB activation, while TIR domain containing adaptor molecule 1 (TICAM1) is necessary for TANK binding kinase 1 (TBK1)-mediated activation of interferon regulatory factor (IRF) transcription factors.</figcaption></figure>

tumor necrosis factor (TNF), through the positive regulation of NF-κB pathways.⁵⁸

In the context of myocardial ischemia-reperfusion injury, a mouse model of coronary artery ligation-induced myocardial damage, ferroptotic cell death initiates neutrophil recruitment and subsequent production of type I interferon (IFN) through the toll like receptor 4 (TLR4) and TIR domain containing adaptor molecule 1 (TICAM1; also known as TRIF) signaling pathway.⁵⁹ This implies that ferroptosis-induced inflammation likely plays a role in the pathogenesis of myocardial ischemia-reperfusion injury. Additionally, 15-hydroperoxyeicosatetraenoic acid, an intermediate metabolite derived from arachidonic acid by ALOX15, acts as a trigger for myocardial ischemia-reperfusion injury by inducing cardiomyocyte ferroptosis.⁶⁰ Both 4HNE and oxidized phospholipids (OxPL) function as ligands for TLR4, leading to the activation of proinflammatory transcription factors, such as NF-κB or the interferon-regulatory factor (IRF) family, via an action on different adaptor proteins.⁶¹

Overall, blocking the signaling pathways activated by DAMPs and PRRs can potentially ameliorate inflammation and tissue damage caused by ferroptotic cell death. Inhibiting the release or recognition of DAMPs, including HMGB1, DCN, 4HNE, and OxPL,

may provide therapeutic benefits by mitigating ferroptosis-induced inflammation. Further investigation is warranted to elucidate the precise mechanisms by which DAMPs released during ferroptosis modulate immune responses and the potential for targeting these pathways as therapeutic strategies in the context of ferroptotic cell death and associated inflammatory diseases.

## 3.2 | Infection by pathogens

Iron plays a crucial role in the proper functioning of immune cells, including macrophages, neutrophils, and lymphocytes.⁶² During infection, the host can regulate iron distribution and availability as a means to modulate immune cell activity. In response to an infection, the body can limit iron availability to pathogens while providing this metal to immune cells, thereby promoting their antimicrobial functions.⁶³ Interestingly, pathogens can exploit iron metabolism to induce ferroptosis in host cells, thereby promoting their propagation or evading host immune surveillance.

For example, *Mycobacterium tuberculosis* can trigger macrophage necrosis associated with iron overload, lipid peroxidation, and downregulation of GPX4 in both in vivo and in vitro models.⁶⁴ In
<figure><img src="image_5.png" /><figcaption>FIGURE 2 Mechanism of ferroptosis-mediated sterile inflammation. Ferroptotic cells can release various damage-associated molecular patterns (DAMPs) that trigger an inflammatory response. For instance, high mobility group box 1 (HMGB1) and decorin (DCN) can bind to advanced glycosylation end-product specific receptor (AGER), activating the nuclear factor kappa B (NF-κB) pathway. This process involves the adaptor protein diaphanous related formin 1 (DIAPH1) and signal transduction through the mitogen-activated protein kinase (MAPK) pathway. Additionally, excessive lipid peroxidation leads to the release of 4-hydroxynonenal (4HNE) and oxidized phospholipids (OxPL). These molecules can activate toll like receptor 4 (TLR4) signaling, resulting in the induction of proinflammatory cytokines (such as interleukin 6 [IL6] and tumor necrosis factor [TNF]) and type I interferon (IFN, such as IFNA and IFNB) through different adaptor proteins and signaling pathways. In general, MYD88 innate immune signal transduction adaptor (MYD88) is required for IKK-dependent NF-κB activation, while TIR domain containing adaptor molecule 1 (TICAM1) is necessary for TANK binding kinase 1 (TBK1)-mediated activation of interferon regulatory factor (IRF) transcription factors.</figcaption></figure>

tumor necrosis factor (TNF), through the positive regulation of NF-κB pathways.⁵⁸

In the context of myocardial ischemia-reperfusion injury, a mouse model of coronary artery ligation-induced myocardial damage, ferroptotic cell death initiates neutrophil recruitment and subsequent production of type I interferon (IFN) through the toll like receptor 4 (TLR4) and TIR domain containing adaptor molecule 1 (TICAM1; also known as TRIF) signaling pathway.⁵⁹ This implies that ferroptosis-induced inflammation likely plays a role in the pathogenesis of myocardial ischemia-reperfusion injury. Additionally, 15-hydroperoxyeicosatetraenoic acid, an intermediate metabolite derived from arachidonic acid by ALOX15, acts as a trigger for myocardial ischemia-reperfusion injury by inducing cardiomyocyte ferroptosis.⁶⁰ Both 4HNE and oxidized phospholipids (OxPL) function as ligands for TLR4, leading to the activation of proinflammatory transcription factors, such as NF-κB or the interferon-regulatory factor (IRF) family, via an action on different adaptor proteins.⁶¹

Overall, blocking the signaling pathways activated by DAMPs and PRRs can potentially ameliorate inflammation and tissue damage caused by ferroptotic cell death. Inhibiting the release or recognition of DAMPs, including HMGB1, DCN, 4HNE, and OxPL,

may provide therapeutic benefits by mitigating ferroptosis-induced inflammation. Further investigation is warranted to elucidate the precise mechanisms by which DAMPs released during ferroptosis modulate immune responses and the potential for targeting these pathways as therapeutic strategies in the context of ferroptotic cell death and associated inflammatory diseases.

## 3.2 | Infection by pathogens

Iron plays a crucial role in the proper functioning of immune cells, including macrophages, neutrophils, and lymphocytes.⁶² During infection, the host can regulate iron distribution and availability as a means to modulate immune cell activity. In response to an infection, the body can limit iron availability to pathogens while providing this metal to immune cells, thereby promoting their antimicrobial functions.⁶³ Interestingly, pathogens can exploit iron metabolism to induce ferroptosis in host cells, thereby promoting their propagation or evading host immune surveillance.

For example, *Mycobacterium tuberculosis* can trigger macrophage necrosis associated with iron overload, lipid peroxidation, and downregulation of GPX4 in both in vivo and in vitro models.⁶⁴ In
<figure><img src="image_6.png" /><figcaption>FIGURE 3 Immune mechanism relevant to ferroptosis-stimulated tumorigenesis. Chronic ferroptotic damage can trigger the release of damage-associated molecular patterns (DAMPs), initiating inflammation-associated immune suppression through multiple mechanisms that promote tumor formation. The release of 8-hydroxyguanosine (8-OHG) from oxidative DNA damage can activate the cyclic GMP-AMP synthase (CGAS)- stimulator of interferon response cGAMP interactor 1 (STING1) pathway, resulting in the production and infiltration of M2 macrophages and myeloid-derived suppressor cells (MDSCs). High mobility group box 1 (HMGB1) can bind to DNA, thereby enhancing CGAS-STING1 pathway activation, and also promoting hypoxia inducible factor 1 subunit alpha (HIF1A)-dependent immune checkpoint CD274 (also known as PD-L1) gene expression. Additionally, oncogenic KRAS<sup>G12D</sup> protein can be released by ferroptotic cancer cells, which, in turn, drives tumor-associated macrophage polarization toward a protumor M2 phenotype through advanced glycosylation end-product specific receptor (AGER).</figcaption></figure>

contrast, treatment with a ferroptosis inhibitor, such as ferrostatin-1, increases the survival of macrophages, as well as their ability to clear the bacteria.⁶⁴ Similarly, in *Pseudomonas aeruginosa* infection, the ALOX15 enzyme catalyzes lipid peroxidation and induces ferroptosis in human bronchial epithelial cells, contributing to lung damage in the context of cystic fibrosis.⁶⁵,⁶⁶ Accordingly, the use of a ferroptosis inhibitor protects against *P. aeruginosa*-induced lung damage in vivo.⁶⁵,⁶⁶ Furthermore, the induction of tumor protein p53 (TP53)-dependent ferroptosis in macrophages during *Listeria* infection leads to massive intracellular iron accumulation, promoting the progression of the infection.⁶⁷ These independent studies suggest that increased ferroptosis in macrophages contributes to the pathogenesis of certain infections. However, in specific circumstances, iron present in macrophages may trigger the oxidative death of intracellular bacteria such as *Staphylococcus aureus* and *Escherichia coli*, hence improving the microbiocidal function of the immune system.⁶⁸

In addition to bacterial infection, dysfunctional ferroptosis has been implicated in viral and parasitic infections. For example, the promotion of certain enteroviruses and coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the strain responsible for causing coronavirus disease 2019 (COVID-19), is facilitated by ACSL4-mediated ferroptosis.⁶⁶ This highlights the potential of developing inhibitors that target ACSL4 as a promising

approach for treating viral diseases. The mechanistic target of rapamycin kinase (MTOR) encompasses two protein complexes, namely MTORC1 and MTORC2, which serve as crucial regulators within the cell's nutrient sensing pathways. MTORC2 deficiency in memory CD4⁺ T cells can induce ferroptosis by suppressing GPX4 activity.⁶⁹ This condition results in heightened vulnerability to acute infection with lymphocytic choriomeningitis virus in mice.⁶⁹ Given that blocking MTOR activation can result in the autophagic degradation of GPX4 protein,⁷⁰ it is essential to further investigate the impact of autophagy on viral infection.

GPX4 is also a selenoenzyme that relies on selenium for its function in preventing lipid ROS-induced ferroptosis. Activation of the selenium-GPX4 axis plays a role in preventing viral infections and enhancing the immune effects of vaccines by inhibiting ferroptosis in follicular helper T cells (T<sub>FH</sub>).⁷¹ Regarding parasitic diseases, infection of *Gpx4*-deficient mice with *Leishmania* leads to a reduction in the number of CD4⁺ T cells, which contributes to the persistence of *Leishmania* in vivo.⁷² On the other hand, the induction of TP53-dependent ferroptosis in liver cells limits infection by the *Plasmodium* parasite.⁷³

The consequences of pathogen infection can lead to the development of sepsis, a clinical syndrome associated with multiple organ dysfunction or failure.⁷⁴ Pharmacological inhibition of ferroptosis
<figure><img src="image_6.png" /><figcaption>FIGURE 3 Immune mechanism relevant to ferroptosis-stimulated tumorigenesis. Chronic ferroptotic damage can trigger the release of damage-associated molecular patterns (DAMPs), initiating inflammation-associated immune suppression through multiple mechanisms that promote tumor formation. The release of 8-hydroxyguanosine (8-OHG) from oxidative DNA damage can activate the cyclic GMP-AMP synthase (CGAS)- stimulator of interferon response cGAMP interactor 1 (STING1) pathway, resulting in the production and infiltration of M2 macrophages and myeloid-derived suppressor cells (MDSCs). High mobility group box 1 (HMGB1) can bind to DNA, thereby enhancing CGAS-STING1 pathway activation, and also promoting hypoxia inducible factor 1 subunit alpha (HIF1A)-dependent immune checkpoint CD274 (also known as PD-L1) gene expression. Additionally, oncogenic KRAS<sup>G12D</sup> protein can be released by ferroptotic cancer cells, which, in turn, drives tumor-associated macrophage polarization toward a protumor M2 phenotype through advanced glycosylation end-product specific receptor (AGER).</figcaption></figure>

contrast, treatment with a ferroptosis inhibitor, such as ferrostatin-1, increases the survival of macrophages, as well as their ability to clear the bacteria.⁶⁴ Similarly, in *Pseudomonas aeruginosa* infection, the ALOX15 enzyme catalyzes lipid peroxidation and induces ferroptosis in human bronchial epithelial cells, contributing to lung damage in the context of cystic fibrosis.⁶⁵,⁶⁶ Accordingly, the use of a ferroptosis inhibitor protects against *P. aeruginosa*-induced lung damage in vivo.⁶⁵,⁶⁶ Furthermore, the induction of tumor protein p53 (TP53)-dependent ferroptosis in macrophages during *Listeria* infection leads to massive intracellular iron accumulation, promoting the progression of the infection.⁶⁷ These independent studies suggest that increased ferroptosis in macrophages contributes to the pathogenesis of certain infections. However, in specific circumstances, iron present in macrophages may trigger the oxidative death of intracellular bacteria such as *Staphylococcus aureus* and *Escherichia coli*, hence improving the microbiocidal function of the immune system.⁶⁸

In addition to bacterial infection, dysfunctional ferroptosis has been implicated in viral and parasitic infections. For example, the promotion of certain enteroviruses and coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the strain responsible for causing coronavirus disease 2019 (COVID-19), is facilitated by ACSL4-mediated ferroptosis.⁶⁶ This highlights the potential of developing inhibitors that target ACSL4 as a promising

approach for treating viral diseases. The mechanistic target of rapamycin kinase (MTOR) encompasses two protein complexes, namely MTORC1 and MTORC2, which serve as crucial regulators within the cell's nutrient sensing pathways. MTORC2 deficiency in memory CD4⁺ T cells can induce ferroptosis by suppressing GPX4 activity.⁶⁹ This condition results in heightened vulnerability to acute infection with lymphocytic choriomeningitis virus in mice.⁶⁹ Given that blocking MTOR activation can result in the autophagic degradation of GPX4 protein,⁷⁰ it is essential to further investigate the impact of autophagy on viral infection.

GPX4 is also a selenoenzyme that relies on selenium for its function in preventing lipid ROS-induced ferroptosis. Activation of the selenium-GPX4 axis plays a role in preventing viral infections and enhancing the immune effects of vaccines by inhibiting ferroptosis in follicular helper T cells (T<sub>FH</sub>).⁷¹ Regarding parasitic diseases, infection of *Gpx4*-deficient mice with *Leishmania* leads to a reduction in the number of CD4⁺ T cells, which contributes to the persistence of *Leishmania* in vivo.⁷² On the other hand, the induction of TP53-dependent ferroptosis in liver cells limits infection by the *Plasmodium* parasite.⁷³

The consequences of pathogen infection can lead to the development of sepsis, a clinical syndrome associated with multiple organ dysfunction or failure.⁷⁴ Pharmacological inhibition of ferroptosis
reduces inflammatory responses and alleviates organ dysfunction.⁷⁵,⁷⁶ Nevertheless, the conditional knockout of *Gpx4* in myeloid cells expedites experimental sepsis triggered by cecal ligation and puncture or bacterial lipopolysaccharides.⁷⁷ This acceleration predominantly occurs through pyroptosis-dependent mechanisms rather than ferroptosis,⁷⁷ emphasizing the fact that lipid peroxidation is also implicated in non-ferroptotic death.⁷⁸ Interestingly, pro-inflammatory M1 macrophages are more resistant to ferroptosis, while anti-inflammatory M2 macrophages are more susceptible to ferroptotic cell death.⁷⁹ Although the underlying mechanisms remain poorly understood, one possibility is that nitric oxide synthase 2 (NOS2; also known as iNOS) is upregulated in M1 macrophages, which may play an antioxidant role to limit ferroptosis.⁷⁹

Understanding the intricate interplay between pathogens and host cells, as well as the activation of antioxidant pathways in response to ferroptotic stress, is of utmost importance in designing approaches to limit infection. By deciphering the mechanisms by which specific immune cell subsets modulate ferroptosis and by exploring the antioxidant pathways that regulate ferroptosis resistance or sensitivity, we can gain insights into potential therapeutic strategies to combat sepsis and other infectious diseases.

## 3.3 | Tumor immunity

The immune system plays a critical role in recognizing and eliminating cancerous cells, contributing to the surveillance and control of tumor growth. Tumor immunity involves a complex interplay of various components and processes that work together to recognize and target tumor cells. Emerging evidence suggests that ferroptotic responses have a multifaceted role in tumor immunity, with implications that vary depending on the tumor stage, model, and treatment context.⁸⁰

While inflammation is a vital part of the immune response against pathogens and tissue injury, chronic or persistent inflammation within the tumor microenvironment can create a favorable environment for tumor development and progression.⁸¹ Particularly, the effects of ferroptotic damage on macrophage polarization, shifting them from an antitumor to a protumor phenotype, have been observed to promote *Kras*-driven pancreatic tumorigenesis in mice (Figure 3).⁸² Induction of ferroptotic damage, either through high-iron diets or depletion of *Gpx4* in pancreatic acinar cells, triggers the release of nuclear DNA containing 8-hydroxy-2'-deoxyguanosine (8-OHG) into the cytosol, subsequently activating the stimulator of interferon response cGAMP interactor 1 (STING1)-dependent DNA sensor pathway.⁸² This, in turn, leads to macrophage infiltration and protumor M2 polarization during *Kras*-driven pancreatic ductal adenocarcinoma development in mice.⁸² Additionally, the release of HMGB1 and oncogenic KRAS protein from ferroptotic cells can promote M2 macrophage polarization in pancreatic cancer or the recruitment and activation of myeloid-derived suppressor cells (MDSCs) in mice with *Gpx4*-deficient liver tumors.⁸³,⁸⁴ Furthermore, HMGB1 can potentiate the cyclic GMP-AMP synthase

(CGAS)-STING1 pathway⁸⁵ and induce the expression of immune checkpoint CD274 (also known as PD-L1) through the activation of hypoxia inducible factor 1 subunit alpha (HIF1A) transcription factor in pancreatic tumor.⁸⁶ These mechanisms contribute to the establishment of an inflammation-associated immune-suppressive tumor microenvironment.

In established tumors, robust induction of ferroptosis has emerged as a promising approach to target tumors,⁸⁷ particularly drug-resistant cells that overexpress GPX4.⁸⁸ One of the advantageous aspects of ferroptosis induction is its potential to activate antitumor immunity by triggering immunogenic cell death (ICD), which is a form of regulated cell death that elicits an immune response and promotes immune system activation.⁸⁹,⁹⁰ Ferroptosis-related ICD is characterized by the release of specific molecular signals, such as adenosine triphosphate (ATP) and HMGB1, as well as the presentation of antigens that alert the immune system to the presence of dying cells (Figure 4).⁹¹ This immune activation can lead to the recognition and elimination of dying cells, as well as associated pathogens or tumor cells.

However, recent studies suggest that ferroptosis can have a negative impact on antigen-presenting cells, such as dendritic cells, impairing the activation of CD8⁺ T cells-mediated adaptive immunity.⁹² The release of immune-suppressive DAMPs during ferroptotic cell death may contribute to this immune suppression.⁹³ While previous studies suggest that oxidized HMGB1 can mediate immune tolerance during apoptosis,⁹⁴ further investigation is needed to determine the redox status of extracellular HMGB1 in the context of ferroptosis. Additionally, the induction of ferroptosis in dendritic cells can directly impair the immunogenicity of ferroptotic cells, potentially dampening the immune response.⁹⁵ Furthermore, the clearance of ferroptotic cells by macrophages may dampen the immunogenicity of these cells. A recent study suggests that toll-like receptor 2 (TLR2) on macrophages can recognize an oxidized phospholipid, specifically 1-stearoyl-2-15-HpETE-sn-glycero-3-phosphatidylethanolamine (SAPE-OOH), on the surface of ferroptotic cells, thereby promoting their phagocytosis.⁹⁶ Gaining deeper insights into the mechanisms underlying the “eat me” and “don’t eat me” signals is crucial for elucidating how immune cells engage in the phagocytosis of ferroptotic cells.

Most current ferroptosis inducers primarily target GPX4 or SLC7A11 in cancer cells, both of which are expressed in immune cells as well. However, the non-selective induction of ferroptosis can have contrasting effects on immune cells within the tumor microenvironment. While ferroptosis can eliminate immunosuppressive cells, such as regulatory T cells ($T_{reg}$)⁹⁷ and MDSCs,⁹⁸ to enhance antitumor immunity, it can also kill immune effectors including CD8⁺ T cells,⁹⁹ natural killer cells,¹⁰⁰ neutrophils,¹⁰¹ and macrophages,⁷⁹ thus dampening antitumor immune responses. Therefore, when evaluating the long-term effects of ferroptosis inducers on tumor immunity in vivo, careful assessment of the diverse immune cell components within the tumor microenvironment is essential. Overcoming this challenge requires the development of strategies that simultaneously inhibit ferroptosis in anti-tumor immune effectors while
reduces inflammatory responses and alleviates organ dysfunction.⁷⁵,⁷⁶ Nevertheless, the conditional knockout of *Gpx4* in myeloid cells expedites experimental sepsis triggered by cecal ligation and puncture or bacterial lipopolysaccharides.⁷⁷ This acceleration predominantly occurs through pyroptosis-dependent mechanisms rather than ferroptosis,⁷⁷ emphasizing the fact that lipid peroxidation is also implicated in non-ferroptotic death.⁷⁸ Interestingly, pro-inflammatory M1 macrophages are more resistant to ferroptosis, while anti-inflammatory M2 macrophages are more susceptible to ferroptotic cell death.⁷⁹ Although the underlying mechanisms remain poorly understood, one possibility is that nitric oxide synthase 2 (NOS2; also known as iNOS) is upregulated in M1 macrophages, which may play an antioxidant role to limit ferroptosis.⁷⁹

Understanding the intricate interplay between pathogens and host cells, as well as the activation of antioxidant pathways in response to ferroptotic stress, is of utmost importance in designing approaches to limit infection. By deciphering the mechanisms by which specific immune cell subsets modulate ferroptosis and by exploring the antioxidant pathways that regulate ferroptosis resistance or sensitivity, we can gain insights into potential therapeutic strategies to combat sepsis and other infectious diseases.

## 3.3 | Tumor immunity

The immune system plays a critical role in recognizing and eliminating cancerous cells, contributing to the surveillance and control of tumor growth. Tumor immunity involves a complex interplay of various components and processes that work together to recognize and target tumor cells. Emerging evidence suggests that ferroptotic responses have a multifaceted role in tumor immunity, with implications that vary depending on the tumor stage, model, and treatment context.⁸⁰

While inflammation is a vital part of the immune response against pathogens and tissue injury, chronic or persistent inflammation within the tumor microenvironment can create a favorable environment for tumor development and progression.⁸¹ Particularly, the effects of ferroptotic damage on macrophage polarization, shifting them from an antitumor to a protumor phenotype, have been observed to promote *Kras*-driven pancreatic tumorigenesis in mice (Figure 3).⁸² Induction of ferroptotic damage, either through high-iron diets or depletion of *Gpx4* in pancreatic acinar cells, triggers the release of nuclear DNA containing 8-hydroxy-2'-deoxyguanosine (8-OHG) into the cytosol, subsequently activating the stimulator of interferon response cGAMP interactor 1 (STING1)-dependent DNA sensor pathway.⁸² This, in turn, leads to macrophage infiltration and protumor M2 polarization during *Kras*-driven pancreatic ductal adenocarcinoma development in mice.⁸² Additionally, the release of HMGB1 and oncogenic KRAS protein from ferroptotic cells can promote M2 macrophage polarization in pancreatic cancer or the recruitment and activation of myeloid-derived suppressor cells (MDSCs) in mice with *Gpx4*-deficient liver tumors.⁸³,⁸⁴ Furthermore, HMGB1 can potentiate the cyclic GMP-AMP synthase

(CGAS)-STING1 pathway⁸⁵ and induce the expression of immune checkpoint CD274 (also known as PD-L1) through the activation of hypoxia inducible factor 1 subunit alpha (HIF1A) transcription factor in pancreatic tumor.⁸⁶ These mechanisms contribute to the establishment of an inflammation-associated immune-suppressive tumor microenvironment.

In established tumors, robust induction of ferroptosis has emerged as a promising approach to target tumors,⁸⁷ particularly drug-resistant cells that overexpress GPX4.⁸⁸ One of the advantageous aspects of ferroptosis induction is its potential to activate antitumor immunity by triggering immunogenic cell death (ICD), which is a form of regulated cell death that elicits an immune response and promotes immune system activation.⁸⁹,⁹⁰ Ferroptosis-related ICD is characterized by the release of specific molecular signals, such asadenosine triphosphate (ATP) and HMGB1, as well as the presentation of antigens that alert the immune system to the presence of dying cells (Figure 4).⁹¹ This immune activation can lead to the recognition and elimination of dying cells, as well as associated pathogens or tumor cells.

However, recent studies suggest that ferroptosis can have a negative impact on antigen-presenting cells, such as dendritic cells, impairing the activation of CD8⁺ T cells-mediated adaptive immunity.⁹² The release of immune-suppressive DAMPs during ferroptotic cell death may contribute to this immune suppression.⁹³ While previous studies suggest that oxidized HMGB1 can mediate immune tolerance during apoptosis,⁹⁴ further investigation is needed to determine the redox status of extracellular HMGB1 in the context of ferroptosis. Additionally, the induction of ferroptosis in dendritic cells can directly impair the immunogenicity of ferroptotic cells, potentially dampening the immune response.⁹⁵ Furthermore, the clearance of ferroptotic cells by macrophages may dampen the immunogenicity of these cells. A recent study suggests that toll-like receptor 2 (TLR2) on macrophages can recognize an oxidized phospholipid, specifically 1-stearoyl-2-15-HpETE-sn-glycero-3-phosphatidylethanolamine (SAPE-OOH), on the surface of ferroptotic cells, thereby promoting their phagocytosis.⁹⁶ Gaining deeper insights into the mechanisms underlying the “eat me” and “don’t eat me” signals is crucial for elucidating how immune cells engage in the phagocytosis of ferroptotic cells.

Most current ferroptosis inducers primarily target GPX4 or SLC7A11 in cancer cells, both of which are expressed in immune cells as well. However, the non-selective induction of ferroptosis can have contrasting effects on immune cells within the tumor microenvironment. While ferroptosis can eliminate immunosuppressive cells, such as regulatory T cells ($T_{reg}$)⁹⁷ and MDSCs,⁹⁸ to enhance antitumor immunity, it can also kill immune effectors including CD8⁺ T cells,⁹⁹ natural killer cells,¹⁰⁰ neutrophils,¹⁰¹ and macrophages,⁷⁹ thus dampening antitumor immune responses. Therefore, when evaluating the long-term effects of ferroptosis inducers on tumor immunity in vivo, careful assessment of the diverse immune cell components within the tumor microenvironment is essential. Overcoming this challenge requires the development of strategies that simultaneously inhibit ferroptosis in anti-tumor immune effectors while
<figure><img src="image_7.png" /><figcaption>FIGURE 4 Immune mechanism of ferroptosis-mediated tumor suppression. Robust ferroptotic damage can trigger the release of damage-associated molecular patterns (DAMPs), initiating immunogenic cell death. The released ATP and high mobility group box 1 (HMGB1) can bind to their respective receptors purinergic receptor P2X 7 (P2RX7) and toll like receptor 4 (TLR4) on immature dendritic cells (DCs), promoting DC maturation and antigen presentation. Subsequently, this process facilitates the cytotoxic T cell response, leading to tumor elimination through the release of cytokines, such as type I interferon (IFN), interleukin 2 (IL2), and interleukin 12 (IL12).</figcaption></figure>

promoting ferroptosis in immunosuppressive cells to maximize the benefits of tumor immunotherapy. At the present stage of research, directly targeting the GPX4 protein is not considered an effective approach for enhancing ferroptosis-related antitumor immunity in vivo. This is primarily due to its potential side effects, including the elimination of immune effectors.

Of note, immune cells can release cytokines that trigger cancer cell death through a ferroptotic mechanism. For example, interferon gamma (IFNG; also known as IFNγ) released from CD8⁺ T cells downregulates the expression of SLC7A11.¹⁰² This downregulation impairs the uptake of cystine by tumor cells, leading to increased lipid peroxidation and subsequent ferroptosis.¹⁰² The combination of locally produced arachidonic acid with IFNG can further enhance IFNG-induced immunogenic ferroptosis of cancer cells, partly through the involvement of ACSL4.¹⁰³ However, in infection models, IFNG can restrict iron uptake by upregulating the expression of the iron exporter solute carrier family 40 member 1 (SLC40A1; also known as FPN1).¹⁰⁴ Thus, IFNG plays a dual role in the regulation of ferroptosis, exerting both restrictive effects on iron intake and potential influences on other aspects of ferroptotic pathways. In contrast, the proinflammatory cytokine interleukin 1 beta (IL1B, also known as IL1β) triggers the translocation of lysine acetyltransferase 2B (KAT2B, also known as PCAF) to mitochondria, where it catalyzes the acetylation of nicotinamide nucleotide transhydrogenase (NNT) at the K1042 site.¹⁰⁵ This acetylation event inhibits ferroptosis and promotes tumor immune evasion by ensuring sufficient maintenance of iron-sulfur clusters.¹⁰⁵ Thus, different cytokines play opposing roles in the regulation of ferroptosis in cancer cells.

Combining ferroptosis inducers with immune checkpoint inhibitors represents another promising approach for treating various cancers. Although preclinical studies are encouraging,¹⁰²,¹⁰⁶,¹⁰⁷

clinical studies investigating this combination are currently lacking. The identification of reliable biomarkers to assess ferroptosis and its correlation with immune response parameters is important for patient stratification and predicting treatment responses in the future. Validated biomarkers could guide personalized therapeutic approaches and help monitor the efficacy of ferroptosis-targeted interventions.¹⁰⁸ For example, a bioinformatics analysis of osteosarcoma patients utilizing the Cancer Genome Atlas database unveiled a 4-gene panel (WASP actin nucleation promoting factor [WAS], cortistatin [CORT], Wnt family member 16 [WNT16], and galactosidase beta 1 like 2 [GLB1L2]) as biomarkers associated with both ferroptosis and immune response in the tumor microenvironment.¹⁰⁹ Furthermore, the redox status of HMGB1 released during cell death, such as ferroptosis, has the potential to shift the antitumor response from immunostimulation to immunosuppression.¹¹⁰

4 | CONCLUSION AND PERSPECTIVE

Over the last 10 years, the field of ferroptosis research has witnessed a remarkable surge in advancements and discoveries.⁴,¹¹¹ It has become evident that ferroptosis influences, and intersects with, various pathological processes traditionally associated with apoptosis or other forms of cell death. This observation suggests the existence of intricate cross-talk and feedback loops among different cell death pathways. Ferroptosis is a distinctive form of oxidative cell death that arises from dysfunctions in multiple antioxidant systems.¹¹² However, the interplay, competition, and complementation between these antioxidant systems in regulating ferroptosis remain poorly understood.
<figure><img src="image_7.png" /><figcaption>FIGURE 4 Immune mechanism of ferroptosis-mediated tumor suppression. Robust ferroptotic damage can trigger the release of damage-associated molecular patterns (DAMPs), initiating immunogenic cell death. The released ATP and high mobility group box 1 (HMGB1) can bind to their respective receptors purinergic receptor P2X 7 (P2RX7) and toll like receptor 4 (TLR4) on immature dendritic cells (DCs), promoting DC maturation and antigen presentation. Subsequently, this process facilitates the cytotoxic T cell response, leading to tumor elimination through the release of cytokines, such as type I interferon (IFN), interleukin 2 (IL2), and interleukin 12 (IL12).</figcaption></figure>

promoting ferroptosis in immunosuppressive cells to maximize the benefits of tumor immunotherapy. At the present stage of research, directly targeting the GPX4 protein is not considered an effective approach for enhancing ferroptosis-related antitumor immunity in vivo. This is primarily due to its potential side effects, including the elimination of immune effectors.

Of note, immune cells can release cytokines that trigger cancer cell death through a ferroptotic mechanism. For example, interferon gamma (IFNG; also known as IFNγ) released from CD8⁺ T cells downregulates the expression of SLC7A11.¹⁰² This downregulation impairs the uptake of cystine by tumor cells, leading to increased lipid peroxidation and subsequent ferroptosis.¹⁰² The combination of locally produced arachidonic acid with IFNG can further enhance IFNG-induced immunogenic ferroptosis of cancer cells, partly through the involvement of ACSL4.¹⁰³ However, in infection models, IFNG can restrict iron uptake by upregulating the expression of the iron exporter solute carrier family 40 member 1 (SLC40A1; also known as FPN1).¹⁰⁴ Thus, IFNG plays a dual role in the regulation of ferroptosis, exerting both restrictive effects on iron intake and potential influences on other aspects of ferroptotic pathways. In contrast, the proinflammatory cytokine interleukin 1 beta (IL1B, also known as IL1β) triggers the translocation of lysine acetyltransferase 2B (KAT2B, also known as PCAF) to mitochondria, where it catalyzes the acetylation of nicotinamide nucleotide transhydrogenase (NNT) at the K1042 site.¹⁰⁵ This acetylation event inhibits ferroptosis and promotes tumor immune evasion by ensuring sufficient maintenance of iron-sulfur clusters.¹⁰⁵ Thus, different cytokines play opposing roles in the regulation of ferroptosis in cancer cells.

Combining ferroptosis inducers with immune checkpoint inhibitors represents another promising approach for treating various cancers. Although preclinical studies are encouraging,¹⁰²,¹⁰⁶,¹⁰⁷

clinical studies investigating this combination are currently lacking. The identification of reliable biomarkers to assess ferroptosis and its correlation with immune response parameters is important for patient stratification and predicting treatment responses in the future. Validated biomarkers could guide personalized therapeutic approaches and help monitor the efficacy of ferroptosis-targeted interventions.¹⁰⁸ For example, a bioinformatics analysis of osteosarcoma patients utilizing the Cancer Genome Atlas database unveiled a 4-gene panel (WASP actin nucleation promoting factor [WAS], cortistatin [CORT], Wnt family member 16 [WNT16], and galactosidase beta 1 like 2 [GLB1L2]) as biomarkers associated with both ferroptosis and immune response in the tumor microenvironment.¹⁰⁹ Furthermore, the redox status of HMGB1 released during cell death, such as ferroptosis, has the potential to shift the antitumor response from immunostimulation to immunosuppression.¹¹⁰

4 | CONCLUSION AND PERSPECTIVE

Over the last 10 years, the field of ferroptosis research has witnessed a remarkable surge in advancements and discoveries.⁴,¹¹¹ It has become evident that ferroptosis influences, and intersects with, various pathological processes traditionally associated with apoptosis or other forms of cell death. This observation suggests the existence of intricate cross-talk and feedback loops among different cell death pathways. Ferroptosis is a distinctive form of oxidative cell death that arises from dysfunctions in multiple antioxidant systems.¹¹² However, the interplay, competition, and complementation between these antioxidant systems in regulating ferroptosis remain poorly understood.
The advancements in our understanding of ferroptosis mechanisms and its immune consequences have had a profound impact on the field of cell biology and hold promise for therapeutic applications in medicine. Modulating ferroptosis and its impact on immune responses may offer opportunities for therapeutic interventions. Combining strategies targeting ferroptosis regulators, such as lipid or iron metabolism pathways, with immunotherapies could enhance anti-tumor immune responses or suppress pathological immune reactions. Given the complex interactions between ferroptosis and immune responses, exploring combination therapies that integrate ferroptosis modulators with immune checkpoint inhibitors, targeted therapies, or other immunomodulatory agents holds potential to improve treatment outcomes.⁸⁰

To propel the exploration of ferroptosis-modulated immune responses forward, future research could focus on the following directions:

1. Immune cell-specific functions: Explore the unique functions of diverse immune cell subsets in the regulation of ferroptosis and their implications for overall immune responses. By comprehending the precise mechanisms through which specific immune cells modulate ferroptosis within specific contexts, we can gain valuable insights into their roles in the development of disease and unveil potential therapeutic approaches.

2. Immune memory and ferroptosis: Explore the influence of ferroptosis on immune memory responses, such as the generation of memory T cells and long-term immune protection. Elucidating the relationship between ferroptosis and immune memory will have implications for vaccine development and the establishment of long-lasting immune control against pathogens or tumors.

3. Translational applications: Utilize the insights obtained from preclinical investigations to advance clinical implementations through the development of therapies aimed at targeting ferroptosis, thereby influencing immune responses. It is essential to carry out clinical trials to assess the effectiveness and safety of combined strategies that involve the modulation of ferroptosis and immunotherapies across various disease contexts.

By pursuing these research directions, we will ultimately succeed in unraveling the intricate interplay between ferroptosis and immune responses. This carries the promise of novel therapeutic strategies that harness the potential of ferroptosis modulation in the context of immune-mediated diseases.

## ACKNOWLEDGMENTS

Research by D.T. and R.K. was supported by grants from the National Institutes of Health (R01CA160417, R01CA229275, and R01CA211070). GK is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR)—Projets blancs; AMMICa US23/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); a donation by Elior; Equipex Onco-Pheno-Screen; European Joint Programme on Rare Diseases (EJPRD); European Research Council Advanced Investigator Award

(ERC-2021-ADG, ICD-Cancer, Grant No. 101052444), European Union Horizon 2020 Projects Oncobiome, Prevalung (grant No. 101095604) and Crimson; Fondation Carrefour; Institut National du Cancer (INCa); Institut Universitaire de France; LabEx Immuno-Oncology (ANR-18-IDEX-0001); a Cancer Research ASPIRE Award from the Mark Foundation; the RHU Immunolife; Seerave Foundation; SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and SIRIC Cancer Research and Personalized Medicine (CARPEM). This study contributes to the IdEx Université de Paris ANR-18-IDEX-0001.

## CONFLICT OF INTEREST STATEMENT

GK has been holding research contracts with Daiichi Sankyo, Eleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Tollys, and Vascage. GK is on the Board of Directors of the Bristol Myers Squibb Foundation France. GK is a scientific co-founder of everImmune, Osasuna Therapeutics, Samsara Therapeutics and Therafast Bio. GK is in the scientific advisory boards of Hevolution, Institut Servier and Longevity Vision Funds. GK is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis and metabolic disorders. GK's wife, Laurence Zitvogel, has held research contracts with Glaxo Smyth Kline, Incyte, Lytix, Kaleido, Innovate Pharma, Daiichi Sankyo, Pilege, Merus, Transgene, 9m, Tusk and Roche, was on the Board of Directors of Transgene, is a cofounder of everImmune, and holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota. GK's brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim. The other authors declare no conflicts of interest or financial interests. The funders had no role in the design of the study; in the writing of the manuscript, or in the decision to publish the results.

## DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

## ORCID

Daolin Tang https://orcid.org/0000-0002-1903-6180

Guido Kroemer https://orcid.org/0000-0002-9334-4405

Rui Kang https://orcid.org/0000-0003-2725-1574

## REFERENCES

1. Galluzzi L, Vitale I, Aaronson SA, et al. Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. *Cell Death Differ.* 2018;25(3):486-541. doi:10.1038/s41418-017-0012-4

2. Tang D, Kang R, Berghe TV, Vandenabeele P, Kroemer G. The molecular machinery of regulated cell death. *Cell Res.* 2019;29(5):347-364. doi:10.1038/s41422-019-0164-5

3. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. *Br J Cancer.* 1972;26(4):239-257. doi:10.1038/bjc.1972.33

4. Stockwell BR. Ferroptosis turns 10: emerging mechanisms, physiological functions, and therapeutic applications. *Cell.* 2022;185(14):2401-2421. doi:10.1016/j.cell.2022.06.003
The advancements in our understanding of ferroptosis mechanisms and its immune consequences have had a profound impact on the field of cell biology and hold promise for therapeutic applications in medicine. Modulating ferroptosis and its impact on immune responses may offer opportunities for therapeutic interventions. Combining strategies targeting ferroptosis regulators, such as lipid or iron metabolism pathways, with immunotherapies could enhance anti-tumor immune responses or suppress pathological immune reactions. Given the complex interactions between ferroptosis and immune responses, exploring combination therapies that integrate ferroptosis modulators with immune checkpoint inhibitors, targeted therapies, or other immunomodulatory agents holds potential to improve treatment outcomes.⁸⁰

To propel the exploration of ferroptosis-modulated immune responses forward, future research could focus on the following directions:

1. Immune cell-specific functions: Explore the unique functions of diverse immune cell subsets in the regulation of ferroptosis and their implications for overall immune responses. By comprehending the precise mechanisms through which specific immune cells modulate ferroptosis within specific contexts, we can gain valuable insights into their roles in the development of disease and unveil potential therapeutic approaches.

2. Immune memory and ferroptosis: Explore the influence of ferroptosis on immune memory responses, such as the generation of memory T cells and long-term immune protection. Elucidating the relationship between ferroptosis and immune memory will have implications for vaccine development and the establishment of long-lasting immune control against pathogens or tumors.

3. Translational applications: Utilize the insights obtained from preclinical investigations to advance clinical implementations through the development of therapies aimed at targeting ferroptosis, thereby influencing immune responses. It is essential to carry out clinical trials to assess the effectiveness and safety of combined strategies that involve the modulation of ferroptosis and immunotherapies across various disease contexts.

By pursuing these research directions, we will ultimately succeed in unraveling the intricate interplay between ferroptosis and immune responses. This carries the promise of novel therapeutic strategies that harness the potential of ferroptosis modulation in the context of immune-mediated diseases.

## ACKNOWLEDGMENTS

Research by D.T. and R.K. was supported by grants from the National Institutes of Health (R01CA160417, R01CA229275, and R01CA211070). GK is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR)—Projets blancs; AMMICa US23/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); a donation by Elior; Equipex Onco-Pheno-Screen; European Joint Programme on Rare Diseases (EJPRD); European Research Council Advanced Investigator Award

(ERC-2021-ADG, ICD-Cancer, Grant No. 101052444), European Union Horizon 2020 Projects Oncobiome, Prevalung (grant No. 101095604) and Crimson; Fondation Carrefour; Institut National du Cancer (INCa); Institut Universitaire de France; LabEx Immuno-Oncology (ANR-18-IDEX-0001); a Cancer Research ASPIRE Award from the Mark Foundation; the RHU Immunolife; Seerave Foundation; SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and SIRIC Cancer Research and Personalized Medicine (CARPEM). This study contributes to the IdEx Université de Paris ANR-18-IDEX-0001.

## CONFLICT OF INTEREST STATEMENT

GK has been holding research contracts with Daiichi Sankyo, Eleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Tollys, and Vascage. GK is on the Board of Directors of the Bristol Myers Squibb Foundation France. GK is a scientific co-founder of everImmune, Osasuna Therapeutics, Samsara Therapeutics and Therafast Bio. GK is in the scientific advisory boards of Hevolution, Institut Servier and Longevity Vision Funds. GK is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis and metabolic disorders. GK's wife, Laurence Zitvogel, has held research contracts with Glaxo Smyth Kline, Incyte, Lytix, Kaleido, Innovate Pharma, Daiichi Sankyo, Pilege, Merus, Transgene, 9m, Tusk and Roche, was on the Board of Directors of Transgene, is a cofounder of everImmune, and holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota. GK's brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim. The other authors declare no conflicts of interest or financial interests. The funders had no role in the design of the study; in the writing of the manuscript, or in the decision to publish the results.

## DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

## ORCID

Daolin Tang https://orcid.org/0000-0002-1903-6180

Guido Kroemer https://orcid.org/0000-0002-9334-4405

Rui Kang https://orcid.org/0000-0003-2725-1574

## REFERENCES

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2. Tang D, Kang R, Berghe TV, Vandenabeele P, Kroemer G. The molecular machinery of regulated cell death. *Cell Res.* 2019;29(5):347-364. doi:10.1038/s41422-019-0164-5

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42. Zhao J, Dar HH, Deng Y, et al. PEBP1 acts as a rheostat between prosurvival autophagy and ferroptotic death cutting-edge asthmatic epithelial cells. *Proc Natl Acad Sci U S A*. 2020;117(25):14376-14385. doi:10.1073/pnas.1921618117

43. Zindel J, Kubes P. DAMPs, PAMPs, and LAMPs in immunity and sterile inflammation. *Annu Rev Pathol*. 2020;15:493-518. doi:10.1146/annurev-pathmechdis-012419-032847

44. Liu K, Liu J, Zou B, et al. Trypsin-mediated sensitization to ferroptosis increases the severity of pancreatitis in mice. *Cell Mol Gastroenterol Hepatol*. 2022;13(2):483-500. doi:10.1016/j.jcmgh.2021.09.008
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49. Su L, Jiang X, Yang C, et al. Pannexin 1 mediates ferroptosis that contributes to renal ischemia/reperfusion injury. *J Biol Chem.* 2019;294(50):19395-19404. doi:10.1074/jbc.RA119.010949

50. Zhang T, Yang M, Ma C, Wei X, Zhang Z. BACH1 encourages ferroptosis by activating KDM4C-mediated COX2 demethylation after cerebral ischemia-reperfusion injury. *Eur J Neurosci.* 2023. doi:10.1111/ejn.16035. Online ahead of print.

51. Rong Y, Fan J, Ji C, et al. USP11 regulates autophagy-dependent ferroptosis after spinal cord ischemia-reperfusion injury by deubiquitinating Beclin 1. *Cell Death Differ.* 2022;29(6):1164-1175. doi:10.1038/s41418-021-00907-8

52. Li Y, Feng D, Wang Z, et al. Ischemia-induced ACSL4 activation contributes to ferroptosis-mediated tissue injury in intestinal ischemia/reperfusion. *Cell Death Differ.* 2019;26(11):2284-2299. doi:10.1038/s41418-019-0299-4

53. Dong H, Qiang Z, Chai D, et al. Nrf2 inhibits ferroptosis and protects against acute lung injury due to intestinal ischemia reperfusion via regulating SLC7A11 and HO-1. *Aging* (Albany NY). 2020;12(13):12943-12959. doi:10.18632/aging.103378

54. Chen Y, Li S, Yin M, et al. Isorhapontigen attenuates cardiac microvascular injury in diabetes via the inhibition of mitochondria-associated ferroptosis through PRDX2-MFN2-ACSL4 pathways. *Diabetes*. 2023;72(3):389-404. doi:10.2337/db22-0553

55. Tsurusaki S, Tsuchiya Y, Koumura T, et al. Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis. *Cell Death Dis.* 2019;10(6):449. doi:10.1038/s41419-019-1678-y

56. Wen Q, Liu J, Kang R, Zhou B, Tang D. The release and activity of HMGB1 in ferroptosis. *Biochem Biophys Res Commun.* 2019;510(2):278-283. doi:10.1016/j.bbrc.2019.01.090

57. Liu J, Zhu S, Zeng L, et al. DCN released from ferroptotic cells ignites AGER-dependent immune responses. *Autophagy*. 2022;18(9):2036-2049. doi:10.1080/15548627.2021.2008692

58. Hudson BI, Lippman ME. Targeting RAGE signaling in inflammatory disease. *Annu Rev Med.* 2018;69:349-364. doi:10.1146/annurev-med-041316-085215

59. Li W, Feng G, Gauthier JM, et al. Ferroptotic cell death and TLR4/Trif signaling initiate neutrophil recruitment after heart transplantation. *J Clin Invest.* 2019;129(6):2293-2304. doi:10.1172/JCI126428

60. Cai W, Liu L, Shi X, et al. Alox15/15-HpETE aggravates myocardial ischemia-reperfusion injury by promoting cardiomyocyte ferroptosis. *Circulation*. 2023;147(19):1444-1460. doi:10.1161/CIRCULATIONAHA.122.060257

61. Imai Y, Kuba K, Neely GG, et al. Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury. *Cell*. 2008;133(2):235-249. doi:10.1016/j.cell.2008.02.043

62. Ni S, Yuan Y, Kuang Y, Li X. Iron metabolism and immune regulation. *Front Immunol.* 2022;13:816282. doi:10.3389/fimmu.2022.816282

63. Paradkar PN, de Domenico I, Durchfort N, Zohn I, Kaplan J, Ward DM. Iron depletion limits intracellular bacterial growth in macrophages. *Blood*. 2008;112(3):866-874. doi:10.1182/blood-2007-12-126854

64. Amaral EP, Costa DL, Namasivayam S, et al. A major role for ferroptosis in *Mycobacterium tuberculosis*-induced cell death and

tissue necrosis. *J Exp Med.* 2019;216(3):556-570. doi:10.1084/jem.20181776

65. Dar HH, Tyurina YY, Mikulska-Ruminska K, et al. *Pseudomonas aeruginosa* utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium. *J Clin Invest.* 2018;128(10):4639-4653. doi:10.1172/JCI9940

66. Ousingsawat J, Schreiber R, Gulbins E, Kamler M, Kunzelmann K. *P. aeruginosa* induced lipid peroxidation causes Ferroptotic cell death in airways. *Cell Physiol Biochem.* 2021;55(5):590-604. doi:10.33594/000000437

67. Singh KS, Leu JI, Barnoud T, et al. African-centric TP53 variant increases iron accumulation and bacterial pathogenesis but improves response to malaria toxin. *Nat Commun.* 2020;11(1):473. doi:10.1038/s41467-019-14151-9

68. Ma R, Fang L, Chen L, Wang X, Jiang J, Gao L. Ferroptotic stress promotes macrophages against intracellular bacteria. *Theranostics.* 2022;12(5):2266-2289. doi:10.7150/thno.66663

69. Wang Y, Tian Q, Hao Y, et al. The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4(+) T cells by preventing ferroptosis. *Nat Immunol.* 2022;23(2):303-317. doi:10.1038/s41590-021-01090-1

70. Liu Y, Wang Y, Liu J, Kang R, Tang D. Interplay between MTOR and GPX4 signaling modulates autophagy-dependent ferroptotic cancer cell death. *Cancer Gene Ther.* 2021;28(1-2):55-63. doi:10.1038/s41417-020-0182-y

71. Yao Y, Chen Z, Zhang H, et al. Selenium-GPX4 axis protects follicular helper T cells from ferroptosis. *Nat Immunol.* 2021;22(9):1127-1139. doi:10.1038/s41590-021-00996-0

72. Matsushita M, Freigang S, Schneider C, Conrad M, Bornkamm GW, Kopf M. T cell lipid peroxidation induces ferroptosis and prevents immunity to infection. *J Exp Med.* 2015;212(4):555-568. doi:10.1084/jem.20140857

73. Kain HS, Glennon EKK, Vijayan K, et al. Liver stage malaria infection is controlled by host regulators of lipid peroxidation. *Cell Death Differ.* 2020;27(1):44-54. doi:10.1038/s41418-019-0338-1

74. Tang D, Wang H, Billiar TR, Kroemer G, Kang R. Emerging mechanisms of immunocoagulation in sepsis and septic shock. *Trends Immunol.* 2021;42(6):508-522. doi:10.1016/j.it.2021.04.001

75. Li N, Wang W, Zhou H, et al. Ferritinophagy-mediated ferroptosis is involved in sepsis-induced cardiac injury. *Free Radic Biol Med.* 2020;160:303-318. doi:10.1016/j.freeradbiomed.2020.08.009

76. Wang Y, Chen D, Xie H, et al. AUF1 protects against ferroptosis to alleviate sepsis-induced acute lung injury by regulating NRF2 and ATF3. *Cell Mol Life Sci.* 2022;79(5):228. doi:10.1007/s00018-022-04248-8

77. Kang R, Zeng L, Zhu S, et al. Lipid peroxidation drives Gasdermin D-mediated Pyroptosis in lethal polymicrobial sepsis. *Cell Host Microbe.* 2018;24(1):97-108.e4. doi:10.1016/j.chom.2018.05.009

83. Dai E, Han L, Liu J, et al. Autophagy-dependent ferroptosis drives tumor-associated macrophage polarization via release and uptake
45. Friedmann Angeli JP, Schneider M, Proneth B, et al. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. *Nat Cell Biol.* 2014;16(12):1180-1191. doi:10.1038/ncb3064

46. Carlson BA, Tobe R, Yefremova E, et al. Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration. *Redox Biol.* 2016;9:22-31. doi:10.1016/j.redox.2016.05.003

47. Mayr L, Grabherr F, Schwarzler J, et al. Dietary lipids fuel GPX4-restricted enteritis resembling Crohn's disease. *Nat Commun.* 2020;11(1):1775. doi:10.1038/s41467-020-15646-6

48. Li P, Jiang M, Li K, et al. Glutathione peroxidase 4-regulated neutrophil ferroptosis induces systemic autoimmunity. *Nat Immunol.* 2021;22(9):1107-1117. doi:10.1038/s41590-021-00993-3

49. Su L, Jiang X, Yang C, et al. Pannexin 1 mediates ferroptosis that contributes to renal ischemia/reperfusion injury. *J Biol Chem.* 2019;294(50):19395-19404. doi:10.1074/jbc.RA119.010949

50. Zhang T, Yang M, Ma C, Wei X, Zhang Z. BACH1 encourages ferroptosis by activating KDM4C-mediated COX2 demethylation after cerebral ischemia-reperfusion injury. *Eur J Neurosci.* 2023. doi:10.1111/ejn.16035. Online ahead of print.

51. Rong Y, Fan J, Ji C, et al. USP11 regulates autophagy-dependent ferroptosis after spinal cord ischemia-reperfusion injury by deubiquitinating Beclin 1. *Cell Death Differ.* 2022;29(6):1164-1175. doi:10.1038/s41418-021-00907-8

52. Li Y, Feng D, Wang Z, et al. Ischemia-induced ACSL4 activation contributes to ferroptosis-mediated tissue injury in intestinal ischemia/reperfusion. *Cell Death Differ.* 2019;26(11):2284-2299. doi:10.1038/s41418-019-0299-4

53. Dong H, Qiang Z, Chai D, et al. Nrf2 inhibits ferroptosis and protects against acute lung injury due to intestinal ischemia reperfusion via regulating SLC7A11 and HO-1. *Aging (Albany NY).* 2020;12(13):12943-12959. doi:10.18632/aging.103378

54. Chen Y, Li S, Yin M, et al. Isorhapontigen attenuates cardiac microvascular injury in diabetes via the inhibition of mitochondria-associated ferroptosis through PRDX2-MFN2-ACSL4 pathways. *Diabetes.* 2023;72(3):389-404. doi:10.2337/db22-0553

55. Tsurusaki S, Tsuchiya Y, Koumura T, et al. Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis. *Cell Death Dis.* 2019;10(6):449. doi:10.1038/s41419-019-1678-y

56. Wen Q, Liu J, Kang R, Zhou B, Tang D. The release and activity of HMGB1 in ferroptosis. *Biochem Biophys Res Commun.* 2019;510(2):278-283. doi:10.1016/j.bbrc.2019.01.090

57. Liu J, Zhu S, Zeng L, et al. DCN released from ferroptotic cells ignites AGER-dependent immune responses. *Autophagy.* 2022;18(9):2036-2049. doi:10.1080/15548627.2021.2008692

58. Hudson BI, Lippman ME. Targeting RAGE signaling in inflammatory disease. *Annu Rev Med.* 2018;69:349-364. doi:10.1146/annurev-med-041316-085215

59. Li W, Feng G, Gauthier JM, et al. Ferroptotic cell death and TLR4/Trif signaling initiate neutrophil recruitment after heart transplantation. *J Clin Invest.* 2019;129(6):2293-2304. doi:10.1172/JCI126428

60. Cai W, Liu L, Shi X, et al. Alox15/15-HpETE aggravates myocardial ischemia-reperfusion injury by promoting cardiomyocyte ferroptosis. *Circulation.* 2023;147(19):1444-1460. doi:10.1161/CIRCULATIONAHA.122.060257

61. Imai Y, Kuba K, Neely GG, et al. Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury. *Cell.* 2008;133(2):235-249. doi:10.1016/j.cell.2008.02.043

62. Ni S, Yuan Y, Kuang Y, Li X. Iron metabolism and immune regulation. *Front Immunol.* 2022;13:816282. doi:10.3389/fimmu.2022.816282

63. Paradkar PN, de Domenico I, Durchfort N, Zohn I, Kaplan J, Ward DM. Iron depletion limits intracellular bacterial growth in macrophages. *Blood.* 2008;112(3):866-874. doi:10.1182/blood-2007-12-126854

64. Amaral EP, Costa DL, Namasivayam S, et al. A major role for ferroptosis in *Mycobacterium tuberculosis*-induced cell death and

tissue necrosis. *J Exp Med.* 2019;216(3):556-570. doi:10.1084/jem.20181776

65. Dar HH, Tyurina YY, Mikulska-Ruminska K, et al. *Pseudomonas aeruginosa* utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium. *J Clin Invest.* 2018;128(10):4639-4653. doi:10.1172/JCI9940

66. Ousingsawat J, Schreiber R, Gulbins E, Kamler M, Kunzelmann K. *P. aeruginosa* induced lipid peroxidation causes Ferroptotic cell death in airways. *Cell Physiol Biochem.* 2021;55(5):590-604. doi:10.33594/000000437

67. Singh KS, Leu JI, Barnoud T, et al. African-centric TP53 variant increases iron accumulation and bacterial pathogenesis but improves response to malaria toxin. *Nat Commun.* 2020;11(1):473. doi:10.1038/s41467-019-14151-9

68. Ma R, Fang L, Chen L, Wang X, Jiang J, Gao L. Ferroptotic stress promotes macrophages against intracellular bacteria. *Theranostics.* 2022;12(5):2266-2289. doi:10.7150/thno.66663

69. Wang Y, Tian Q, Hao Y, et al. The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4(+) T cells by preventing ferroptosis. *Nat Immunol.* 2022;23(2):303-317. doi:10.1038/s41590-021-01090-1

70. Liu Y, Wang Y, Liu J, Kang R, Tang D. Interplay between MTOR and GPX4 signaling modulates autophagy-dependent ferroptotic cancer cell death. *Cancer Gene Ther.* 2021;28(1-2):55-63. doi:10.1038/s41417-020-0182-y

71. Yao Y, Chen Z, Zhang H, et al. Selenium-GPX4 axis protects follicular helper T cells from ferroptosis. *Nat Immunol.* 2021;22(9):1127-1139. doi:10.1038/s41590-021-00996-0

72. Matsushita M, Freigang S, Schneider C, Conrad M, Bornkamm GW, Kopf M. T cell lipid peroxidation induces ferroptosis and prevents immunity to infection. *J Exp Med.* 2015;212(4):555-568. doi:10.1084/jem.20140857

73. Kain HS, Glennon EKK, Vijayan K, et al. Liver stage malaria infection is controlled by host regulators of lipid peroxidation. *Cell Death Differ.* 2020;27(1):44-54. doi:10.1038/s41418-019-0338-1

74. Tang D, Wang H, Billiar TR, Kroemer G, Kang R. Emerging mechanisms of immunocoagulation in sepsis and septic shock. *Trends Immunol.* 2021;42(6):508-522. doi:10.1016/j.it.2021.04.001

75. Li N, Wang W, Zhou H, et al. Ferritinophagy-mediated ferroptosis is involved in sepsis-induced cardiac injury. *Free Radic Biol Med.* 2020;160:303-318. doi:10.1016/j.freeradbiomed.2020.08.009

76. Wang Y, Chen D, Xie H, et al. AUF1 protects against ferroptosis to alleviate sepsis-induced acute lung injury by regulating NRF2 and ATF3. *Cell Mol Life Sci.* 2022;79(5):228. doi:10.1007/s00018-022-04248-8

77. Kang R, Zeng L, Zhu S, et al. Lipid peroxidation drives Gasdermin D-mediated Pyroptosis in lethal polymicrobial sepsis. *Cell Host Microbe.* 2018;24(1):97-108.e4. doi:10.1016/j.chom.2018.05.009

82. Dai E, Han L, Liu J, et al. Ferroptotic damage promotes pancreatic tumorigenesis through a TMEM173/STING-dependent DNA sensor pathway. *Nat Commun.* 2020;11(1):6339. doi:10.1038/s41467-020-20154-8

83. Dai E, Han L, Liu J, et al. Autophagy-dependent ferroptosis drives tumor-associated macrophage polarization via release and uptake
of oncogenic KRAS protein. *Autophagy*. 2020;16(11):2069-2083.
doi:10.1080/15548627.2020.1714209

84. Conche C, Finkelmeier F, Pesic M, et al. Combining ferroptosis induction with MDSC blockade renders primary tumours and metastases in liver sensitive to immune checkpoint blockade. *Gut*. 2023;gutjnl-2022-327909. doi:10.1136/gutjnl-2022-327909.
Online ahead of print.

85. Andreeva L, Hiller B, Kostrewa D, et al. cGAS senses long and HMGB/TFAM-bound U-turn DNA by forming protein-DNA ladders. *Nature*. 2017;549(7672):394-398. doi:10.1038/nature23890

86. Li C, Zhang Y, Cheng X, et al. PINK1 and PARK2 suppress pancreatic tumorigenesis through control of mitochondrial iron-mediated immunometabolism. *Dev Cell*. 2018;46(4):441-455.e8.
doi:10.1016/j.devcel.2018.07.012

87. Chen X, Kang R, Kroemer G, Tang D. Broadening horizons: the role of ferroptosis in cancer. *Nat Rev Clin Oncol*. 2021;18(5):280-296.
doi:10.1038/s41571-020-00462-0

88. Hangauer MJ, Viswanathan VS, Ryan MJ, et al. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. *Nature*. 2017;551(7679):247-250. doi:10.1038/nature24297

89. Galluzzi L, Vitale I, Warren S, et al. Consensus guidelines for the definition, detection and interpretation of immunogenic cell death. *J Immunother Cancer*. 2020;8(1):e000337. doi:10.1136/jitc-2019-000337

90. Kroemer G, Galassi C, Zitvogel L, Galluzzi L. Immunogenic cell stress and death. *Nat Immunol*. 2022;23(4):487-500. doi:10.1038/s41590-022-01132-2

91. Efimova I, Catanzaro E, van der Meeren L, et al. Vaccination with early ferroptotic cancer cells induces efficient antitumor immunity. *J Immunother Cancer*. 2020;8(2):e001369. doi:10.1136/jitc-2020-001369

92. Wiernicki B, Maschalidi S, Pinney J, et al. Cancer cells dying from ferroptosis impede dendritic cell-mediated anti-tumor immunity. *Nat Commun*. 2022;13(1):3676. doi:10.1038/s41467-022-31218-2

93. Hayashi K, Nikolos F, Lee YC, et al. Tipping the immunostimulatory and inhibitory DAMP balance to harness immunogenic cell death. *Nat Commun*. 2020;11(1):6299. doi:10.1038/s41467-020-19970-9

94. Kazama H, Ricci JE, Herndon JM, Hoppe G, Green DR, Ferguson TA. Induction of immunological tolerance by apoptotic cells requires caspase-dependent oxidation of high-mobility group box-1 protein. *Immunity*. 2008;29(1):21-32. doi:10.1016/j.immuni.2008.05.013

95. Han L, Bai L, Qu C, et al. PPARG-mediated ferroptosis in dendritic cells limits antitumor immunity. *Biochem Biophys Res Commun*. 2021;576:33-39. doi:10.1016/j.bbrc.2021.08.082

96. Luo X, Gong HB, Gao HY, et al. Oxygenated phosphatidylethanolamine navigates phagocytosis of ferroptotic cells by interacting with TLR2. *Cell Death Differ*. 2021;28(6):1971-1989. doi:10.1038/s41418-020-00719-2

97. Xu C, Sun S, Johnson T, et al. The glutathione peroxidase Gpx4 prevents lipid peroxidation and ferroptosis to sustain Treg cell activation and suppression of antitumor immunity. *Cell Rep*. 2021;35(11):109235. doi:10.1016/j.celrep.2021.109235

98. Zhu H, Klement JD, Lu C, et al. Asah2 represses the p53-Hmox1 Axis to protect myeloid-derived suppressor cells from ferroptosis. *J Immunol*. 2021;206(6):1395-1404. doi:10.4049/jimmunol.2000500

99. Xu S, Chaudhary O, Rodriguez-Morales P, et al. Uptake of oxidized lipids by the scavenger receptor CD36 promotes lipid peroxidation and dysfunction in CD8(+) T cells in tumors. *Immunity*. 2021;54(7):1561-1577.e7. doi:10.1016/j.immuni.2021.05.003

100. Poznanski SM, Singh K, Ritchie TM, et al. Metabolic flexibility determines human NK cell functional fate in the tumor microenvironment. *Cell Metab*. 2021;33(6):1205-1220.e5. doi:10.1016/j.cmet.2021.03.023

101. Kim R, Hashimoto A, Markosyan N, et al. Ferroptosis of tumour neutrophils causes immune suppression in cancer. *Nature*. 2022;612(7939):338-346. doi:10.1038/s41586-022-05443-0

102. Wang W, Green M, Choi JE, et al. CD8(+) T cells regulate tumour ferroptosis during cancer immunotherapy. *Nature*. 2019;569(7755):270-274. doi:10.1038/s41586-019-1170-y

103. Liao P, Wang W, Wang W, et al. CD8(+) T cells and fatty acids orchestrate tumor ferroptosis and immunity via ACSL4. *Cancer Cell*. 2022;40(4):365-378.e6. doi:10.1016/j.ccell.2022.02.003

104. Nairz M, Fritsche G, Brunner P, Talasz H, Hantke K, Weiss G. Interferon-gamma limits the availability of iron for intramacrophage salmonella typhimurium. *Eur J Immunol*. 2008;38(7):1923-1936. doi:10.1002/eji.200738056

105. Han Y, Zhang YY, Pan YQ, et al. IL-1beta-associated NNT acetylation orchestrates iron-sulfur cluster maintenance and cancer immunotherapy resistance. *Mol Cell*. 2023;83(11):1887-1902.e8. doi:10.1016/j.molcel.2023.05.011

106. Cheu JW, Lee D, Li Q, et al. Ferroptosis suppressor protein 1 inhibition promotes tumor ferroptosis and anti-tumor immune responses in liver cancer. *Cell Mol Gastroenterol Hepatol*. 2023;16(1):133-159. doi:10.1016/j.jcmgh.2023.03.001

107. Jiang Z, Lim SO, Yan M, et al. TYRO3 induces anti-PD-1/PD-L1 therapy resistance by limiting innate immunity and tumoral ferroptosis. *J Clin Invest*. 2021;131(8):e139434. doi:10.1172/JCI139434

108. Chen X, Comish PB, Tang D, Kang R. Characteristics and biomarkers of ferroptosis. *Front Cell Dev Biol*. 2021;9:637162. doi:10.3389/fcell.2021.637162

109. Li G, Lei J, Xu D, Yu W, Bai J, Wu G. Integrative analyses of ferroptosis and immune related biomarkers and the osteosarcoma associated mechanisms. *Sci Rep*. 2023;13(1):5770. doi:10.1038/s41598-023-33009-1

110. Chen R, Zou J, Kang R, Tang D. The redox protein HMGB1 in cell death and cancer. *Antioxid Redox Signal*. 2023. doi:10.1089/ars.2023.0007. Online ahead of print.

111. Tang D, Kang R. From oxytosis to ferroptosis: 10 years of research on oxidative cell death. *Antioxid Redox Signal*. 2023. doi:10.1089/ars.2023.0356. Online ahead of print.

112. Kuang F, Liu J, Tang D, Kang R. Oxidative damage and antioxidant defense in ferroptosis. *Front Cell Dev Biol*. 2020;8:586578. doi:10.3389/fcell.2020.586578

**How to cite this article:** Tang D, Kroemer G, Kang R. Ferroptosis in immunostimulation and immunosuppression. *Immunol Rev*. 2024;321:199-210. doi:10.1111/imr.13235
of oncogenic KRAS protein. *Autophagy*. 2020;16(11):2069-2083.
doi:10.1080/15548627.2020.1714209

84. Conche C, Finkelmeier F, Pesic M, et al. Combining ferroptosis induction with MDSC blockade renders primary tumours and metastases in liver sensitive to immune checkpoint blockade. *Gut*. 2023;gutjnl-2022-327909. doi:10.1136/gutjnl-2022-327909.
Online ahead of print.

85. Andreeva L, Hiller B, Kostrewa D, et al. cGAS senses long and HMGB/TFAM-bound U-turn DNA by forming protein-DNA ladders. *Nature*. 2017;549(7672):394-398. doi:10.1038/nature23890

86. Li C, Zhang Y, Cheng X, et al. PINK1 and PARK2 suppress pancreatic tumorigenesis through control of mitochondrial iron-mediated immunometabolism. *Dev Cell*. 2018;46(4):441-455.e8. doi:10.1016/j.devcel.2018.07.012

87. Chen X, Kang R, Kroemer G, Tang D. Broadening horizons: the role of ferroptosis in cancer. *Nat Rev Clin Oncol*. 2021;18(5):280-296. doi:10.1038/s41571-020-00462-0

88. Hangauer MJ, Viswanathan VS, Ryan MJ, et al. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. *Nature*. 2017;551(7679):247-250. doi:10.1038/nature24297

89. Galluzzi L, Vitale I, Warren S, et al. Consensus guidelines for the definition, detection and interpretation of immunogenic cell death. *J Immunother Cancer*. 2020;8(1):e000337. doi:10.1136/jitc-2019-000337

90. Kroemer G, Galassi C, Zitvogel L, Galluzzi L. Immunogenic cell stress and death. *Nat Immunol*. 2022;23(4):487-500. doi:10.1038/s41590-022-01132-2

91. Efimova I, Catanzaro E, van der Meeren L, et al. Vaccination with early ferroptotic cancer cells induces efficient antitumor immunity. *J Immunother Cancer*. 2020;8(2):e001369. doi:10.1136/jitc-2020-001369

92. Wiernicki B, Maschalidi S, Pinney J, et al. Cancer cells dying from ferroptosis impede dendritic cell-mediated anti-tumor immunity. *Nat Commun*. 2022;13(1):3676. doi:10.1038/s41467-022-31218-2

93. Hayashi K, Nikolos F, Lee YC, et al. Tipping the immunostimulatory and inhibitory DAMP balance to harness immunogenic cell death. *Nat Commun*. 2020;11(1):6299. doi:10.1038/s41467-020-19970-9

94. Kazama H, Ricci JE, Herndon JM, Hoppe G, Green DR, Ferguson TA. Induction of immunological tolerance by apoptotic cells requires caspase-dependent oxidation of high-mobility group box-1 protein. *Immunity*. 2008;29(1):21-32. doi:10.1016/j.immuni.2008.05.013

95. Han L, Bai L, Qu C, et al. PPARG-mediated ferroptosis in dendritic cells limits antitumor immunity. *Biochem Biophys Res Commun*. 2021;576:33-39. doi:10.1016/j.bbrc.2021.08.082

96. Luo X, Gong HB, Gao HY, et al. Oxygenated phosphatidylethanolamine navigates phagocytosis of ferroptotic cells by interacting with TLR2. *Cell Death Differ*. 2021;28(6):1971-1989. doi:10.1038/s41418-020-00719-2

97. Xu C, Sun S, Johnson T, et al. The glutathione peroxidase Gpx4 prevents lipid peroxidation and ferroptosis to sustain Treg cell activation and suppression of antitumor immunity. *Cell Rep*. 2021;35(11):109235. doi:10.1016/j.celrep.2021.109235

98. Zhu H, Klement JD, Lu C, et al. Asah2 represses the p53-Hmox1 Axis to protect myeloid-derived suppressor cells from ferroptosis. *J Immunol*. 2021;206(6):1395-1404. doi:10.4049/jimmunol.2000500

99. Xu S, Chaudhary O, Rodriguez-Morales P, et al. Uptake of oxidized lipids by the scavenger receptor CD36 promotes lipid peroxidation and dysfunction in CD8(+) T cells in tumors. *Immunity*. 2021;54(7):1561-1577.e7. doi:10.1016/j.immuni.2021.05.003

100. Poznanski SM, Singh K, Ritchie TM, et al. Metabolic flexibility determines human NK cell functional fate in the tumor microenvironment. *Cell Metab*. 2021;33(6):1205-1220.e5. doi:10.1016/j.cmet.2021.03.023

101. Kim R, Hashimoto A, Markosyan N, et al. Ferroptosis of tumour neutrophils causes immune suppression in cancer. *Nature*. 2022;612(7939):338-346. doi:10.1038/s41586-022-05443-0

102. Wang W, Green M, Choi JE, et al. CD8(+) T cells regulate tumour ferroptosis during cancer immunotherapy. *Nature*. 2019;569(7755):270-274. doi:10.1038/s41586-019-1170-y

103. Liao P, Wang W, Wang W, et al. CD8(+) T cells and fatty acids orchestrate tumor ferroptosis and immunity via ACSL4. *Cancer Cell*. 2022;40(4):365-378.e6. doi:10.1016/j.ccell.2022.02.003

104. Nairz M, Fritsche G, Brunner P, Talasz H, Hantke K, Weiss G. Interferon-gamma limits the availability of iron for intramacrophage salmonella typhimurium. *Eur J Immunol*. 2008;38(7):1923-1936. doi:10.1002/eji.200738056

105. Han Y, Zhang YY, Pan YQ, et al. IL-1beta-associated NNT acetylation orchestrates iron-sulfur cluster maintenance and cancer immunotherapy resistance. *Mol Cell*. 2023;83(11):1887-1902.e8. doi:10.1016/j.molcel.2023.05.011

106. Cheu JW, Lee D, Li Q, et al. Ferroptosis suppressor protein 1 inhibition promotes tumor ferroptosis and anti-tumor immune responses in liver cancer. *Cell Mol Gastroenterol Hepatol*. 2023;16(1):133-159. doi:10.1016/j.jcmgh.2023.03.001

107. Jiang Z, Lim SO, Yan M, et al. TYRO3 induces anti-PD-1/PD-L1 therapy resistance by limiting innate immunity and tumoral ferroptosis. *J Clin Invest*. 2021;131(8):e139434. doi:10.1172/JCI139434

108. Chen X, Comish PB, Tang D, Kang R. Characteristics and biomarkers of ferroptosis. *Front Cell Dev Biol*. 2021;9:637162. doi:10.3389/fcell.2021.637162

109. Li G, Lei J, Xu D, Yu W, Bai J, Wu G. Integrative analyses of ferroptosis and immune related biomarkers and the osteosarcoma associated mechanisms. *Sci Rep*. 2023;13(1):5770. doi:10.1038/s41598-023-33009-1

110. Chen R, Zou J, Kang R, Tang D. The redox protein HMGB1 in cell death and cancer. *Antioxid Redox Signal*. 2023. doi:10.1089/ars.2023.0007. Online ahead of print.

111. Tang D, Kang R. From oxytosis to ferroptosis: 10 years of research on oxidative cell death. *Antioxid Redox Signal*. 2023. doi:10.1089/ars.2023.0356. Online ahead of print.

112. Kuang F, Liu J, Tang D, Kang R. Oxidative damage and antioxidant defense in ferroptosis. *Front Cell Dev Biol*. 2020;8:586578. doi:10.3389/fcell.2020.586578

**How to cite this article:** Tang D, Kroemer G, Kang R. Ferroptosis in immunostimulation and immunosuppression. *Immunol Rev*. 2024;321:199-210. doi:10.1111/imr.13235