Relationship between ferroptosis and mitophagy in acute lung injury: a mini-review

Introduction

Acute lung injury (ALI), a prevalent and detrimental disorder of the respiratory system, is typified by neutrophil migration through the epithelial cell interstitium accompanied by an unchecked inflammatory response. This leads to substantial damage to lung epithelial cells and disruption of the cellular barrier, with the more severe stages evolving into acute respiratory distress syndrome (ARDS) (Butt, Kurdowska & Allen, 2016; Long, Mallampalli & Horowitz, 2022). Although earlier etiological control (Mokrá, 2020) and modified fluid management (Lee, Corl & Levy, 2021) have reduced its incidence, ALI continues to be associated with substantial morbidity and a high mortality rate. Accounting for over 10% of intensive care unit admissions and 4% of all hospitalizations, the elucidation of ALI and ARDS pathogenesis is pivotal to enhancing treatment for ALI (Meyer, Gattinoni & Calfee, 2021).

The primary function of the lungs is to carry out gas exchange, maintaining the ventilation-perfusion ratio within the normal range in the circulatory system. The energy required for this process is primarily supplied by the mitochondria (Hu & Königshoff, 2022). However, when mitochondrial function is impaired, it leads to an incomplete reduction of O₂, subsequently producing reactive oxygen species (ROS) (Willems et al., 2015). Excessive ROS leads to a switch in cellular energy production from aerobic glycolysis to anaerobic glycolysis and pentose phosphate pathway, affecting the antioxidant capacity of the mitochondria (Schumacker et al., 2014). This further induces mitochondrial damage and the release of mitochondrial DNA (mtDNA), thereby activating inflammatory responses leading to ALI (Schumacker et al., 2014; Long et al., 2022). Persistent mitochondrial damage and dysfunction result in organ failure and poor outcomes in patients with ALI (Ten & Ratner, 2020). Therefore, the removal of unhealthy mitochondria and the generation of healthy mitochondria via mitochondrial quality control (MQC) are critically important for preserving the structural and functional integrity of mitochondria (Ng, Wai & Simonsen, 2021). Mitophagy, a selective autophagic process, is crucial for maintaining mitochondrial quantity, quality, and essential functions within cells (Ashrafi & Schwarz, 2013). This process facilitates the removal of damaged mitochondria and mitigates ROS production, thereby playing a vital role in the occurrence and development of ALI.

Ferroptosis is a newly recognized type of cell death that is involved in the onset of ALI (Liu, Zhang & Xie, 2022; Ma et al., 2023; Wang et al., 2023c). As an increasingly recognized therapeutic target, the inhibition of ferroptosis presents a promising strategy for mitigating ALI (Zhang et al., 2022). Despite growing interest, a thorough understanding of ferroptosis is still evolving. Ferroptosis is often accompanied by the accumulation of large amounts of iron ions and excessive lipid peroxidation products, characterized morphologically by alterations in the ultrastructure of mitochondria including reduced volume, heightened double-membrane density, and disruption of the outer mitochondrial membrane (Li et al., 2020b). During ferroptosis, the redox balance in the cell is disrupted, with levels of glutathione (GSH) and glutathione peroxidase 4 (GPX4) decreasing while ROS levels begin to increase (Jiang, Stockwell & Conrad, 2021). Mitochondria, as the main source of ROS in cells, are tightly associated to ferroptosis (Wang et al., 2020b; Javadov, 2022). Mitophagy facilitates the removal of damaged mitochondria, prevents mitochondrial dysfunction, and thus maintains normal mitochondrial morphology and functionality (Pickles, Vigié & Youle, 2018). However, the relationship between mitophagy and ferroptosis is currently unclear. Current research mainly focuses on the individual roles of ferroptosis and mitophagy in ALI (Liu, Zhang & Xie, 2022; Tang et al., 2023). However, there are fewer studies on the relationship between ferroptosis and mitophagy, especially their correlation in ALI is yet to be investigated. Consequently, in the context of ALI, exploring the direct or indirect interplay between mitophagy and ferroptosis is hypothesized to be fruitful, and dissecting their mechanisms could yield significant insights. In this review, we hypothesize that elucidating the relationship between mitophagy and ferroptosis will aid in finding effective treatment strategies for ALI. We aim to investigate and examine the disturbance mechanism between ferroptosis and mitophagy in ALI by exploring their interrelationship. This review delves into the novel interrelationship between mitophagy and ferroptosis in ALI, aiming to illuminate potential therapeutic avenues for this condition.

Survey methodology

The literature search was conducted in PubMed, Web of Science, and the China National Knowledge Infrastructure database. In addition to considering articles published since 2010, earlier articles were also taken into account. The following keywords were used: ALI, ARDS, ferroptosis, mitophagy, ALI and ferroptosis, ALI and mitophagy, iron metabolism, ferroptosis mechanisms, mitophagy mechanisms. As our work progressed, we conducted literature searches using the keywords NLRP3 and mitophagy, NLRP3 and ferroptosis, mTOR and mitophagy, mTOR and ferroptosis. After removing duplicate articles and the articles with little relevance, 126 articles were selected for inclusion in this review.

Acute lung injury and mitophagy

ALI is often accompanied by mitochondrial damage and dysfunction. To maintain homeostasis, lung tissue cells regulate the number of mitochondria they have to meet their physiological needs by undergoing processes like mitochondrial fission, fusion, and mitophagy (Dutra Silva et al., 2021). Normally, mitophagy is required to maintain cellular equilibrium through the decomposition and degradation of impaired mitochondria. Therefore, an elevated degree of mitophagy may result in mitochondrial malfunction, cellular impairment, and eventual cell death (Chan, 2020).

Regulation mechanism of mitophagy

Mitophagy regulation falls into two primary categories: non-receptor-mediated and receptor-mediated. Non-receptor-mediated mitophagy mainly involves the PINK1 (PTEN-induced kinase 1)/Parkin pathway, while receptor-mediated mitophagy includes proteins such as FUN14 domain containing 1 (FUNDC1), BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), Bcl-2 homology 3 (BH3)-only protein Nix (BNIP3L), and Bcl2 family proteins. Should the PINK1/Parkin pathway be compromised, the receptor-mediated pathway may serve a compensatory function.

PINK1/Parkin and mitophagy

The PINK1/Parkin pathway is the foremost mechanism responsible for mediating mitophagy and represents a well-studied pathway in this context (Tanaka, 2020). PINK1, a serine/threonine kinase present on mitochondria, operates in concert with Parkin, an E3 ubiquitin ligase residing in the cytosol. In normal circumstances, PINK1 moves to the mitochondrial inner membrane (MIM) driven by the mitochondrial transmembrane potential (Δφ), and its mitochondrial targeting signal at the N-terminus is cut by the mitochondrial processing protease (MPP) (Fig. 1A) (Sekine & Youle, 2018). In the hydrophobic region of the inner membrane, the presenilin-associated rhomboid-like protein (PARL) cleaves PINK1 between amino acids Ala-103 (A103) and Phe-104 (F104), leading to the return of the residual PINK1 with F104 to the cytoplasm for rapid degradation via the ubiquitin proteasome system (UPS) (Deas et al., 2011). When mitochondria become damaged, the mitochondrial membrane potential decreases, preventing PINK1 from being degraded in the MIM. Consequently, PINK1 accumulates on the mitochondrial outer membrane (MOM), recruiting and phosphorylating Parkin to ubiquitinate various mitochondrial protein substrates (Kane et al., 2014). These ubiquitinated proteins bind to autophagy microtubule-associated protein 1, light chain 3 (LC3) to initiate mitophagy (Fig. 1B) (Geisler et al., 2010).

FUNDC1 receptor and mitophagy

FUNDC1 is a MOM protein that harbors an LC3 interacting region (LIR) motif, enabling its interaction with LC3 amid hypoxic or mitochondrial stress conditions to facilitate mitophagy (Yang et al., 2019). Under normal physiological circumstances, FUNDC1 is present in a phosphorylated state. During hypoxic episodes, FUNDC1 is dephosphorylated, enhancing its binding to LC3 to promote mitophagy (Liu et al., 2012; Chen et al., 2014). This phosphorylation and dephosphorylation are regulated by the sarcoma gene (Src) kinase and creatine kinase 2 (CK2). Src kinase and CK2 can phosphorylate FUNDC1 at Ser13 and Tyr18 sites under normal physiological conditions, resulting in the formation of inactive p-FUNDC1 and the inhibition of mitophagy (Zheng et al., 2022). Under hypoxic conditions, Src kinase and CK2 remove phosphate groups from FUNDC1, increasing its binding with LC3 to start the process of mitophagy (Wang et al., 2020c). The stimulation of mitophagy via FUNDC1 is additionally mediated by ubiquitination. Under hypoxic conditions, the ubiquitin ligase MARCH5 catalyzes the ubiquitination of FUNDC1 at the Lys119 location, consequently driving the degradation of FUNDC1 (Fig. 1C) (Chen et al., 2017).

BCL-2 protein family and mitophagy

Members of the BCL-2 protein family are key regulators of cell apoptosis, with roles in regulating mitochondrial metabolism and dynamics. Bcl2 like 13 (BCL2L13) receptor, BNIP3, and BNIP3L/NIX, which are part of the BCL-2 protein family, have LIR domains that engage with LC3 to trigger mitophagy (Rogov et al., 2017; Li et al., 2020c). The mitophagy-inducing capacity of these proteins is modulated by their phosphorylation status. Phosphorylation of Ser272 in BCL2L13 enhances its binding to LC3, thereby strengthening its ability to induce mitophagy (Fig. 1C) (Murakawa et al., 2015). Under hypoxic conditions, c-Jun N-terminal kinase 1/2 (JNK 1/2) phosphorylates BNIP3 at Ser60/Thr66. This modification not only prevents the degradation of BNIP3 degradation by the proteosome, but also increases its stability, promotes LC3 interaction, and facilitates mitophagy. Conversely, dephosphorylation of BNIP3 by protein phosphatase 1/2 A (PP1/2A) leads to its proteasomal degradation, thereby impeding mitophagy (Fig. 1D) (He et al., 2022a). BNIP3L collaborates with the Atg8 family of proteins to direct autophagosomes to the intended mitochondria. BNIP3L/NIX primarily utilizes three domains to mediate mitophagy: LIR, the transmembrane (TM) domain and the BH3 domain. The transmembrane domain ensures localization of BNIP3L to the mitochondria, while the interplay between the LIR domain and LC3 can instigate mitophagy. Phosphorylation of NIX at Ser34/35 can augment mitophagy by promoting the interaction between the LIR and LC3, in addition to attracting mitochondria-specific autophagosomes (Rogov et al., 2017). Moreover, the BH3 domain at the NIX amino terminal holds the capacity to associate with Bcl-2, causing the dissociation of the Bcl-2/Beclin-1 complex and subsequent release of Beclin-1, thereby facilitating autophagosome formation and initiating mitophagy (Fig. 1D) (Bellot et al., 2009).

Mitochondrial fission and fusion and mitophagy

Mitochondria are dynamic organelles undergoing constant fission and fusion. Mitochondrial fission plays a crucial role in quality control, as it can divide damaged mitochondria into two: one with normal function and one with impaired function (Ni, Williams & Ding, 2015). DRP1 mediates mitochondrial fission through its action on mitochondrial fission factor (MFF), Mitochondrial Fission 1 protein (Fis-1), and mitochondrial dynamics proteins of 49 and 51 kDa (Mid49/51) (Losón et al., 2013). Subsequently, mitophagy maintains mitochondrial homeostasis by removing dysfunctional mitochondria (Fig. 1E) (Meyer, Leuthner & Luz, 2017). An imbalance in the processes of fission and fusion—a condition that results in mitochondrial fragmentation—serves as a prerequisite for mitophagy (Chan, 2020). Mitophagy is facilitated by mitochondrial fission, and conversely, suppression of mitophagy is achieved through the inhibition of mitochondrial fission or the stimulation of mitochondrial fusion (Wang et al., 2020a).

Acute lung injury and ferroptosis

Ferroptosis, first identified in RAS mutant tumor cells upon erastin exposure, exhibits unique morphological characteristics distinct from apoptosis, necrosis, and other forms of cell death (Dolma et al., 2003). This process is marked by mitochondrial shrinkage, diminution or loss of mitochondrial cristae, condensed mitochondrial membranes, and moderate chromatin condensation (Li et al., 2020b). Subsequent research has elucidated the role of ferroptosis in the pathogenesis of a myriad of diseases, including cancer (Lei, Zhuang & Gan, 2022), lung (Wang et al., 2022a) and kidney disorders (Wang et al., 2022b), as well as myocardial ischemia-reperfusion injury (Cai et al., 2023). In the context of pulmonary conditions, ferroptosis has been linked to ALI triggered by various factors such as infection, radiation, ischemia-reperfusion, drowning, and oleic acid exposure (Table 1). In ALI, disturbed iron homeostasis leads to iron accumulation in the lower respiratory tract, precipitating ferroptosis, which, in concert with oxidative stress and mitochondrial dysfunction, aggravates lung damage (Fig. 2).

Ferroptosis plays a crucial role in the occurrence and progression of sepsis related ALI (SRALI). In a mouse model of ALI caused by LPS, there is a notable rise in the levels of unbound iron in the bronchial epithelial cells of ALI mice, accompanied by a significant decrease in the levels of ferroptosis indicators GPX4 and solute carrier family 7, membrane 11 (SLC7A11). Administering ferrostatin-1 as a preventive measure significantly reduced the severity of ALI, highlighting the crucial involvement of ferroptosis in the development of LPS-induced ALI. Additionally, research has shown that the P53/SLC7A11 and Nrf2/ARE pathways could play a role in controlling ferroptosis in LPS-induced ALI. It has been observed that STAT6 can suppress ferroptosis and reduce ALI by adjusting the P53/SLC7A11 pathway (Yu et al., 2014; Liu et al., 2020; Yang et al., 2022). Ferroptosis also plays a significant role in radiation related ALI (RRALI). In an RRALI mouse model, a decrease in GPX4 expression—a marker of ferroptosis—and mitochondrial alterations suggestive of ferroptosis were visible under transmission electron microscopy. Following treatment with an ferroptosis inhibitor, levels of lung ROS and serum inflammatory factors (TNF-α, IL-6, IL-10, and TGF-β1) significantly decrease, indicating the critical role of ferroptosis in RRALI (Li, Zhuang & Qiao, 2019). Li et al. (2022a) have revealed that activating the P62-Keap1-Nrf2 pathway could prevent radiation-induced ferroptosis in RRALI.

In ischemia-reperfusion related ALI (IIRALI), the cellular tumor antigen p53 modulates disease progression by curbing apoptosis and ferroptosis. Li et al. (2020a) showed that the inhibitor of apoptosis stimulating p53 protein (iASPP) can suppress ferroptosis and alleviate ALI induced by intestinal ischemia-reperfusion in mice by mediating the Nrf2/HIF-1/TF pathway (Li et al., 2020a). Additionally, isoliquiritigenin shields against this ALI by diminishing HIF-1-associated ferroptosis (Zhongyin et al., 2022). In cases of drowning related ALI (DRALI), employing an Nrf2-specific activator, such as dimethyl fumarate, results in the reduction of ROS and lipid ROS levels, a rise in GPX4 mRNA levels, and preservation of Δφ. Additionally, Nrf2 knockout mice show more pronounced lung damage than wild-type mice, suggesting that Nrf2 can suppress ferroptosis and mitigate ALI caused by seawater exposure (Qiu et al., 2020). Ling (2022) further showed that in seawater drowning-induced ALI, SOX9 facilitates lung epithelial cell ferroptosis via the TNFAIP3-ACSL4 pathway. In a murine model of oleic acid-related ALI (OARALI), reduced levels of GPX4 and ferritin are observed along with GSH depletion and accumulation of malondialdehyde in lung tissues, indicating the presence of ferroptosis (Chen et al., 2019; Zhou et al., 2019). These discoveries underscore ferroptosis as a key factor in ALI pathogenesis and a potential therapeutic target, warranting deeper examination into its specific pathways.

Ferroptosis and mitophagy in acute lung injury

The pathogenesis of ALI is closely related to mitophagy and ferroptosis, indicating potential interactions between them. Recent studies have unveiled significant interactions between ferroptosis and mitophagy: mitophagy can regulate the process of cellular ferroptosis (Table 2) (Bi et al., 2024). When cells undergo pathological changes, dysfunctional mitochondria produce excessive ROS and release pro-apoptotic factors, leading to further cell damage. Therefore, removal of dysfunctional mitochondria is essential for cellular homeostasis and survival. Ferroptosis leads to pathological alterations in cells, and mitophagy can clear dysfunctional mitochondria (Liu et al., 2023). Interestingly, similar to the role of mitophagy in ALI, it can both augment and suppress ferroptosis. In the early stage, mitophagy may sequester iron in autophagosomes, reducing the source of ROS in ferroptosis. However, in the massive mitophagy stage, it could provide additional iron, thus increasing lipid peroxidation and ferroptosis (Yu et al., 2022). Therefore, depending on the degree of mitophagy, its effects on ferroptosis may differ.

Ferroptosis and mitophagy can interact through multiple network nodes, which can in turn influence each other and play unique roles. For instance, O-GlcNAcylation (a major nutrient sensor of the glucose flux) can regulate mitophagy and ferroptosis through ferritinophagy. Yu et al. (2022) found that O-GlcNAcylation promotes ferritinophagy, regulates mitophagy, and thereby controls ferroptosis. When ferroptosis inducers induce cell ferroptosis, the O-glucosyltransferase is deactivated, leading to de-O-GlcNAcylation, consequently activating ferritinophagy and mitophagy; ferritinophagy and mitophagy together provide ferrous ions, leading to the rapid generation of ROS and lipid peroxidation, ultimately resulting in ferroptosis. Moreover, inhibiting mitophagy by knocking down PINK1 at least partially prevents de-O-GlcNAcylation-promoted ferroptosis, and simultaneously inhibiting these two pathways nearly completely prevents ferroptosis (Yu et al., 2022). Song et al. (2024) also found ferritinophagy and mitophagy to play a crucial role in cell ferroptosis. In the process of Hexavalent chromium-induced cell death, ferritinophagy increases, disrupting iron homeostasis and releasing ferrous ions. In addition, hexavalent chromium triggers mitophagy, thereby releasing additional ferrous ions. These two pathways induce the simultaneous release of ferrous ions, thereby intensifying lipid peroxidation and ultimately triggering ferroptosis in DF-1 cells (Song et al., 2024).

In conclusion, there are close connections between mitophagy and ferroptosis, and a deeper exploration of the related mechanisms can provide new insights for research on ALI, which is closely related to mitophagy and ferroptosis. However, the relationship between mitophagy-related ferroptosis and ALI has not yet been studied. We aim to identify a common network node among these three by analyzing the relationships between mitophagy and ALI, and ferroptosis and ALI, as well as the regulatory mechanism of mitophagy on ferroptosis. From a mechanistic perspective, we found that both mTOR and NLRP3 play significant roles in the regulation of mitophagy and ferroptosis (Fig. 3).

Conclusions

In this review, we report that both mitophagy and ferroptosis are closely related to the onset and development of ALI. Mitophagy degrades damaged or dysfunctional mitochondria through multiple regulatory mechanisms, and in turn maintains the quality control of mitochondria and regulates ROS released by damaged mitochondria, thus playing an important role in ALI. Secondly, it is worth noting that ferroptosis is also closely related to ROS, mitophagy and ALI, and studies on both provide potential therapeutic options for ALI. However, current research is still insufficient, as the functions of mitophagy vary under different conditions in relation to ALI and ferroptosis, possibly depending on its regulatory mechanisms, extent and different causes of injury, further in-depth research is needed. Lastly, current studies often focus on either inhibiting ferroptosis or independently regulating mitophagy; therefore, the mutual regulation mechanisms between mitophagy and ferroptosis in ALI need further exploration. Through our investigation and analysis, in ALI, NLRP3 and mTOR play a significant ‘bridging’ role between ferroptosis and mitophagy, providing promising new targets for in-depth studies on the molecular mechanisms and interactions involved in these two processes. In summary, there is a close relationship between mitophagy, ferroptosis and ALI, with NLRP3 and mTOR acting as key nodes within these relationships. A deeper understanding of these regulatory processes could provide new insights into ALI research.

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