MLKL regulates intracellular trafficking of neutrophil elastase to promote lung epithelial cell senescence

Abstract

Neutrophil elastase (NE) is a key contributor to the pathogenesis of chronic obstructive pulmonary disease (COPD), driving airway inflammation and tissue destruction. While the receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis has been implicated in cigarette smoke-induced COPD, its role in NE-mediated lung injury remains undefined. In this study, we demonstrate that NE does not induce necroptosis in lung epithelial cells in vitro. Although genetic deletion of RIPK3 or MLKL did not prevent NE-induced apoptosis, the absence of MLKL significantly reduced NE-induced cellular senescence. Mechanistically, we found that after NE is internalized into endosomes, MLKL facilitates its escape into the cytoplasm and subsequent translocation to the nucleus, where it induces DNA damage and senescence. In contrast, MLKL deficiency retains NE within endosomes, promoting its degradation via lysosomal trafficking. Furthermore, Mlkl knockout mice were protected from porcine pancreatic elastase-induced emphysema, highlighting the pathological relevance of this mechanism. Collectively, our findings reveal a novel, necroptosis-independent function of MLKL in regulating intracellular NE trafficking, suggesting a new therapeutic target for protease-driven lung injury in COPD.

Introduction

Chronic obstructive pulmonary disease (COPD), one of the significant healthcare problems in the world today, is a chronic lung condition characterized by persistent airflow limitation and inflammation in the lung.¹ Pulmonary emphysema is a major component of COPD, characterized by the permanent enlargement of air spaces due to a process of alveolar destruction and loss of elastic recoil.² Cigarette smoking (CS) is a major risk factor for COPD, which triggers an inflammatory response in the lungs, leading to the recruitment and activation of neutrophils and the release of neutrophil elastase (NE).^3,4 This elastase, in turn, damages the lung tissue, contributing to the development of emphysema and other COPD symptoms.³ Previous studies have shown that CS exposure leads to lung epithelial cell necroptosis,^5-7 an inflammatory form of programmed cell death, which contributes to chronic inflammation in the lungs and thereby exacerbates tissue damage and the development of emphysema.⁶ However, it remains unclear whether NE in the inflammatory lung environment can induce lung epithelial cell necroptosis in the pathogenesis of COPD.

The necroptotic signaling induced by CS in the lung epithelial cells is mainly modulated by the receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL).^6,7 RIPK3 induces necroptosis by phosphorylating and activating MLKL, which then translocates to the cell membrane and initiates cell lysis, characteristic of necroptosis.^8,9 Depletion or inhibition of RIPK3 can protect mice from emphysema when exposed to long-term CS.^6,7 However, it is also unknown whether RIPK3 and MLKL contribute to the lung tissue damage caused by inflammatory NE secretion. NE is a major inflammatory protease released by neutrophils and is present in the airways of patients with COPD.¹⁰ Although NE facilitates leukocyte transmigration to the site of infection and is required for the clearance of Gram-negative bacteria,¹¹ excess NE interrupts lung epithelial cell repair by promoting cellular death,¹² activating inflammation directly by increasing cytokine expression and release,¹³ as well as indirectly by triggering the release of extracellular traps.¹⁴ NE has been reported to induce apoptosis, senescence, and autophagy in lung epithelial cells.^15-17 Despite these findings, the involvement of RIPK3 and MLKL in NE-induced lung injury remains largely undefined.

In this study, we investigate the functional roles of RIPK3 and MLKL in NE-induced lung epithelial cell damage using RIPK3 KO and MLKL KO mice and lung epithelial cells. Our data showed that the absence of RIPK3 or MLKL did not suppress NE-induced cell death. However, MLKL depletion compromised NE-induced senescence by suppressing intracellular NE translocation into the nucleus. Mechanically, we found that the absence of MLKL reduced endosome release of NE and enhanced the translocation of endosomal NE into lysosomes, where NE can be degraded. Our data revealed a novel function of MLKL in the intracellular transportation of NE, which is necroptosis-independent.

Materials and methods

Cell culture and reagents

The Beas-2B human lung epithelial cells, purchased from American Type Culture Collection (Manassas, VA), were cultured in DMEM/F12 (Lonza) containing 5% FBS and 1% Penicillin/Streptomycin, or FBS-free BEGM conditional medium (Lonza).

Mouse lung primary organoids containing airway and alveolar cell types were generated following established protocols.¹⁸ Briefly, the single cells from distal lung tissue were resuspended with 40 μL of Corning Matrigel (Corning, 354230) and cultured with medium (Advanced DEME/F12 with 50% L-WRN cells conditioned media, including B27, TGF-β receptor inhibitor, antioxidants, p38 MAPK inhibitor, FGF-7, FGF-10, and ROCK inhibitor).

Bafilomycin A1 (BafA1, R&D Systems) was dissolved in DMSO (Sigma-Aldrich) for a stock solution. The human NE (Elastin Products Inc.) and porcine pancreatic elastase (PPE, Sigma-Aldrich) were dissolved in PBS for stock solutions.

Cell viability, cell cycle analysis, and senescence assays

Cell viability assay was performed using the MTS assay (Promega, Madison, WI), crystal violet staining (Sigma-Aldrich), and FITC-Annexin-V/7-AAD staining (Invitrogen), followed by flow cytometry (BD FACSverse) as previously described.⁶

For cell cycle analysis, cells were fixed with 70% ethanol, stained with propidium iodide (PI, Invitrogen), and analyzed by flow cytometry (BD FACSverse). The cell cycle stages were quantified by FlowJo (BD Biosciences).

Senescence was evaluated by SA-β-Gal staining (Cell Signaling) following fixation in 2% formaldehyde/0.2% glutaraldehyde and overnight incubation in X-gal solution at 37 °C according to the manufacturer’s instructions.

Western blotting and immunofluorescence

Western blotting was conducted as previously described.¹⁹ The following antibodies were used: RIPK3, human p-RIPK3 (S227), human p-MLKL (S358), MLKL, NE (Abcam), IRS1, Lamin B, cleaved caspase 3, GAPDH, EEA1, P21, and β-actin (Cell Signaling).

For immunofluorescence, fixed cells and tissue sections (5 µm) were incubated with primary antibodies overnight at 4 °C, including MLKL, NE (Abcam), LAMP1 (Santa Cruz Biotechnology), P21, cleaved caspase-3, and EEA1 (Cell Signaling), followed by fluorophore-conjugated secondary antibodies and DAPI staining. Images were acquired using a Leica TCS SP8 confocal microscope. Image quantification was performed using Image J.

CRISPR/Cas9 transfection and subcellular fraction

Guide RNAs targeting MLKL and RIPK3 were cloned into pSpCas9(BB)-2A-GFP (Addgene #48138) as previously described.¹⁹ Nuclear fractions were isolated using the NE-PER kit (ThermoFisher), and endosomes using the Endosome Isolation Kit (GenTex). NE activity in nuclear fractions was measured using EnzChek Elastase Assay Kit (ThermoFisher) and normalized to total protein.

Animal studies

All animal experiments were performed according to the Institutional Animal Care and Use Committee of the University of Southern California. The Ripk3 KO and Mlkl KO mice were gifts from Dr. Newton Kim (Genentech, CA) and Dr. James Murphy (WEHI Research Institute, Australia). The 6 to 8-week-old C57BL/6J mice were treated with intranasal instillation (I.N.) of either 4 μg/kg of PPE (n = 7) or PBS (saline control) (n = 5). After 28 days, lung mechanics were assessed using FlexiVent (SCIREQ).^20,21 Lungs were processed for histology, western blot, and bronchoalveolar lavage (BAL). Alveolar chord length was quantified using Scion Image software (Scion Corp.).²²

Statistical analysis

Data were analyzed using GraphPad Prism with Student’s t-test; P < .05 was considered significant. Results are presented as mean ± SD.

Results

MLKL contributed to NE-induced lung epithelial cell damage

To test if RIPK3 and MLKL are involved in NE-induced lung epithelial cell damage, we generated RIPK3 KO and MLKL KO Beas-2B cells as previously described.¹⁹ We then treated the wild type (WT), RIPK3 KO, and MLKL KO Beas-2B cells with varying doses of NE for 16 h. Our data showed that NE treatment suppressed cell viability in a dosage-dependent manner in Beas-2B cells (Figure 1A). Depletion of RIPK3 didn’t recover the cell viability suppressed by NE treatment (Figure 1A and B). Although the absence of MLKL failed to protect Beas-2B cells from loss of cell viability caused by high dosage of NE (40 nM) (Figure 1A and B), it can partially rescue the cell viability upon the low dosages of NE (10 and 20 nM) treatment (Figure 1A). The long-term NE exposure (10 nM) suppressed cell growth, as shown by colony formation (Figure 1C and D), and this suppression was compromised in MLKL KO cells, but not in RIPK3-depleted cells (Figure 1C and D). To further confirm the effect of RIPK3 and MLKL, we used lung organoids derived from Ripk3 KO and Mlkl KO mice. Similarly, 20 nM NE suppressed the viability of WT mouse lung organoids, and this effect was alleviated in the Mlkl-deficient lung organoids, rather than the Ripk3-deficient lung organoids (Figure 1E and F). These data indicate that MLKL, rather than RIPK3, may be involved in NE function in lung epithelial cells.

Figure 1.

MLKL is required for NE-induced suppression of cell viability. (A) The WT, RIPK3 KO, and MLKL KO Beas-2B cells were exposed to different doses of NE in the FBS-free conditional medium for 16 h. The cell viability was analyzed using the MTS assay (N = 3). (B) The WT, RIPK3 KO, and MLKL KO Beas-2B cells were exposed to 40 nM NE in the FBS-free conditional medium for 16 h. The cell viability was analyzed using crystal violet staining. (C and D) The WT, RIPK3 KO, and MLKL KO Beas-2B cells were seeded in a 6-well plate (1000/well) and exposed to 10 nM NE in the FBS-free conditional medium for 7 days. (C) The colony formation was counted after staining with crystal violet. (D) The representative picture was presented. (E and F) The WT, Ripk3 KO, and Mlkl KO lung organoids were exposed to 20 nM NE in the FBS-free conditional medium for 16 h. (E) The cell viability was analyzed using the MTS assay (N = 3). (F) The representative picture of an organoid was presented. Scale bar, 50 μm. Each experiment was repeated at least 3 times. P (t-test), *P < .05; NS, P > .05.

MLKL depletion suppressed NE-induced lung epithelial cell senescence

Since RIPK3 and MLKL are well-known necroptosis modulators,^5,19 we first determined whether NE-induced suppression of cell viability is due to necroptosis by analyzing the necroptosis markers, phosphorylated RIPK3 (p-RIPK3) and phosphorylated MLKL (p-MLKL), in Beas-2B cells following NE treatment. Our data showed that NE treatment didn’t induce phosphorylation of RIPK3 and MLKL in Beas-2B (Figure 2A), with 8% cigarette smoking extract (CSE)-treated Beas-2B cells as a positive control.⁶ In contrast, the apoptosis and senescence signals were found in the NE-treated Beas-2B (Figure 2A), as indicated by cleavage of caspase-3 and induction of P21 (Figure 2A). The flow cytometry analysis further confirmed that NE administration induced apoptosis in Beas-2B cells (Figure 2B). However, depletion of MLKL didn’t protect the Beas-2B from NE-induced apoptosis (Figure 2B), nor did it suppress the cleavage of caspase-3 (Figure 2C). Considering that NE has been reported to cause senescence by inducing DNA damage,^23,24 we also tested whether MLKL is involved. Our data showed that NE treatment increased the number of ruptured nuclei, which were absent in the MLKL KO cells (Figure 2D). NE treatment also changed the cell cycle distribution by shifting from G0/1 to G2/M in Beas-2B cells (Figure 2E), which was also abolished by MLKL depletion (Figure 2E). However, depletion of MLKL didn’t affect the sub-G population changes in Beas-2B cells upon 20 nM NE treatment (Figure 2E), further confirming that MLKL depletion was not involved in NE-induced apoptosis. The P21 and DNA damage marker, γH2AX, were induced by NE in WT Beas-2B cells, which was compromised in the MLKL KO Beas-2B cells (Figure 2F). Furthermore, NE treatment also enhanced β-gal activity in Beas-2B and mouse lung organoids, which was compromised by MLKL depletion (Figure 2G and H). These data collectively indicated that MLKL mediated NE-induced DNA damage and senescence, which is independent of its function in necroptosis.

Figure 2.

MLKL depletion suppressed NE-induced lung epithelial cell senescence. (A) Western blot of indicated proteins in WT Beas-2B cells treated with 20 nM NE or 8% CSE for 16 h. (B-F) The WT and MLKL KO Beas-2B cells were treated with different doses of NE in the FBS-free conditional medium for 16 h. (B) Flow cytometry analysis for the cells stained with Annexin-V/7-AAD. (C) The western blot of the indicated proteins in different groups of cells. (D) The DAPI staining of the nucleus in the WT and MLKL KO Beas-2B cells treated with 20 nM NE for 16 h. Scale bar, 10 μm. (E) Flow cytometry analysis for the ethanol-fixed cells stained with PI. Left, the representative pictures and the percentage of G2/M phase were shown; right, the quantification of cell population in each cell cycle stage. (F) The western blot of the indicated proteins in different groups of cells. (G) The SA-β-Gal staining of the WT and MLKL KO Beas-2B cells treated with 20 nM NE for 16 h. Scale bar, 20 μm. (H) The SA-β-Gal staining of the WT and Mlkl KO mouse lung organoids treated with 20 nM NE for 16 h. Scale bar, 40 μm. Each experiment was repeated at least 3 times. P (t-test), *P < .05; NS, P > .05.

Absence of MLKL protects against DNA damage by suppressing intracellular NE content

NE can be transported into the nucleus and contribute to chromatin decondensation by proteolysis of histones,²⁵ thereby causing DNA damage.¹² We next examined whether MLKL has a role in NE intracellular trafficking and nuclear accumulation. Immunofluorescence analysis showed that MLKL depletion significantly reduced NE accumulation in the nuclei of Beas-2B cells, as demonstrated by NE immunofluorescence in the nuclei (Figure 3A) and western blot of NE in the nuclear fractionation (Figure 3B). These findings were further confirmed by nuclear fractionation assays followed by NE activity analysis to quantify the NE content in the nuclei (Figure 3C). Additionally, total intracellular NE levels were decreased in MLKL-deficient cells following NE exposure (Figure 3D), suggesting that MLKL contributes to intracellular NE retention. To explore the functional consequence of this reduction in NE levels, we analyzed the expression of IRS1, a known intracellular substrate targeted for degradation by NE.²⁶ Our results demonstrated that MLKL depletion protected IRS1 from degradation following NE exposure (Figure 3D), indicating that MLKL plays a crucial role in maintaining intracellular NE levels necessary for IRS1 cleavage. In contrast, RIPK3 depletion did not exhibit a similar protective effect on IRS1 degradation (Figure 3E), suggesting that this regulatory mechanism is specific to MLKL. To further assess whether MLKL affects NE uptake or clearance, we measured intracellular NE levels over time before and after removal of NE-containing medium. NE accumulation peaked approximately 1.5 h after exposure, followed by a gradual decline as cells cleared NE after medium removal (Figure 3F). Interestingly, MLKL depletion did not affect the initial accumulation of NE within the first 1.5 h (Figure 3G), indicating that MLKL does not regulate NE uptake. However, after medium removal, intracellular NE levels declined more rapidly in MLKL knockout cells compared to controls (Figure 3G), suggesting that MLKL deficiency promotes NE clearance. Collectively, these findings suggest that MLKL facilitates NE nuclear translocation by maintaining intracellular NE content, likely through limiting NE degradation. In the absence of MLKL, enhanced NE clearance reduces nuclear trafficking and downstream proteolytic activity, such as IRS1 degradation.

Figure 3.

Absence of MLKL protects against DNA damage by suppressing intracellular NE content. (A) The WT and MLKL KO Beas-2B cells were treated with 20 nM NE in the FBS-free conditional medium for 2 h. The NE nucleus localization was analyzed by immunofluorescence staining. Scale bar, 5 μm. (B-D) The WT and MLKL KO Beas-2B cells were treated with 10 and 20 nM NE in the FBS-free conditional medium for 4 h. (B) The NE in the nucleus was analyzed with subcellular fractionation followed by western blot. (C) The NE in the nucleus was quantified by measuring the NE activity in the nucleus fractionation of 20 nM NE-treated cells. (D) The expression of indicated proteins in the total cell lysates was analyzed by western blot. Left, the representative blots; right, the quantification of indicated proteins. (E) The WT and RIPK3 KO Beas-2B cells were treated with 10 and 20 nM NE in the FBS-free conditional medium for 4 h. The expression of indicated proteins was analyzed by western blot. (F) The WT Beas-2B cells were treated with 20 nM NE in the FBS-free conditional medium (NE+). Two hours later, the medium was replaced to remove NE (NE−). The accumulation of NE in the cells at the indicated time points was analyzed by western blot. (G) The WT and MLKL KO Beas-2B cells were treated as in (F). The accumulation of NE in the cells at the indicated time points was analyzed by western blot. Each experiment was repeated at least three times. P (t-test), *P < .05; **P < .01; ***P < .001.

MLKL accumulation in the endosome promotes NE release into the cytoplasm

A previous study showed that after entering into EEA-1⁺endosomes, NE either traffics to LAMP-1⁺lysosomes or “leaks” into the cytosol.²⁷ MLKL has been implicated in regulating endosomal trafficking, particularly by facilitating lipid processing in macrophages.²⁸ In the present study, we found that MLKL begins to accumulate in EEA1⁺endosomes in Beas-2B cells as early as 30 min following NE exposure, with sustained presence observed at 60 min (Figure 4A). Consistent with this, NE accumulation within EEA1⁺endosomes also progressively increased over the 60-min exposure period (Figure 4B). Upon removal of NE-containing medium for 15 min, NE levels within endosomes decreased in WT cells (Figure 4B). Interestingly, MLKL depletion resulted in enhanced co-localization of NE with EEA1⁺endosomes and impaired the reduction of endosomal NE levels following NE medium removal (Figure 4B), suggesting that MLKL facilitates NE trafficking out of the endosomal compartment. This observation was further confirmed by endosome subcellular fractionation followed by western blotting, which showed increased NE accumulation in endosomes of MLKL-deficient cells compared to WT controls (Figure 4C). Given that endosomes typically fuse with lysosomes to facilitate cargo degradation, we next assessed NE trafficking to lysosomes. In WT Beas-2B cells, a portion of internalized NE was delivered to LAMP1⁺lysosomes (Figure 4D). However, MLKL depletion resulted in greater NE accumulation within lysosomes (Figure 4D), indicating impaired NE escape from the endosomal-lysosomal pathway. To further evaluate the role of lysosomal degradation in regulating intracellular NE levels, we treated both WT and MLKL knockout Beas-2B cells with the lysosomal inhibitor BafA1. Inhibition of lysosomal function with BafA1 abolished the effects of MLKL deficiency on both NE accumulation and IRS1 degradation (Figure 4E), confirming that lysosomal degradation is the primary mechanism responsible for NE clearance when MLKL is absent. Collectively, these findings suggest that MLKL translocation to endosomes facilitates NE escape from the endosomal-lysosomal degradation pathway, thereby limiting NE degradation and allowing its accumulation in the cytosol.

Figure 4.

MLKL accumulation in the endosome promotes NE release into the cytoplasm. (A) The WT Beas-2B cells were treated with 20 nM NE in the FBS-free conditional medium for 30 and 60 min. The accumulation of MLKL in endosomes was analyzed by immunofluorescent staining for MLKL and EEA1. Scale bar, 5 μm. (B-D) The WT and MLKL KO Beas-2B cells were treated with 20 nM NE in the FBS-free conditional medium. After 60 min, the medium was replaced to remove NE. (B) The accumulation of NE in the endosome was analyzed by immunofluorescent staining of NE and EEA1 at the indicated time points. Scale bar, 10 μm. (C) The accumulation of NE in the endosome and cytosol was analyzed by western blot. (D) The accumulation of NE in the lysosome was analyzed by immunofluorescent staining of NE and LAMP1 at 30 min after NE removal. Scale bar, 10 μm. (E) The WT and MLKL KO Beas-2B cells were pretreated with BafA1 (50 nM) for 1 h and treated with 20 nM NE in the FBS-free conditional medium for an additional 2 h. The expression of indicated proteins was analyzed by western blot. Each experiment was repeated at least 3 times.

Depletion of MLKL protects against PPE-induced emphysema

Intratracheal administration of PPE, a porcine homolog of NE, in animals has been reported to reproduce key phenomena of emphysema.⁴ To further confirm the role of MLKL in NE-induced lung injury in vivo, we treated WT and Ripk3, Mlkl KO C57BL/6J mice with PPE via I.N. administration. WT mice I.N. administered with PPE had significant airspace enlargement compared with PBS controls (Figure 5A and B). Depletion of Ripk3 didn’t protect against the alveolar enlargement caused by PPE I.N. treatment (Figure 5A and B). In contrast, Mlkl absence attenuated the PPE I.N.-induced alveolar enlargement (Figure 5A and B). Administration of PPE suppressed the tissue elastance (H) and increased the neutrophil number in BAL (Figure 5C and D). Depletion of Mlkl also restored lung tissue elastance and suppressed the number of neutrophils in the BAL (Figure 5C and D). Therefore, our results suggested that MLKL depletion suppressed NE-induced emphysema in vivo.

Figure 5.

Mlkl KO mice had less emphysema upon PPE instillation. The 6 to 8-weeks old WT, Ripk3 KO, and Mlkl KO C57BL/6 mice were treated with 4 μg/kg PPE via I.N. instillation. Four weeks later, the lung tissues were dissected. (A) Representative pictures of the lung tissue gill staining. Scale bar, 40 μm. (B) Chord lengths of mouse lungs in the indicated groups were calculated. (C) The tissue elastance (H) of lung tissues was analyzed by FlexinVent. (D) The neutrophil number in the BAL from different groups of mice. P (t-test), *P < .05; **P < .01; *** P < .001; NS, P > .05.

MLKL depletion suppressed the NE burden in vivo

To further investigate the functional role of MLKL in NE in vivo, we analyzed NE levels in WT and Mlkl KO mice with or without PPE I.N. administration. Our data showed that PPE I.N. administration enhanced the NE level in the lung tissue of WT mice (Figure 6A). Depletion of Mlkl suppressed the NE level in the lung tissue upon PPE treatment (Figure 6A). Consistent with the data in vitro, we also observed enhanced cleavage of caspase-3 in the WT lung tissue upon PPE treatment, which was not affected by the Mlkl KO (Figure 6A). The immunofluorescence staining of cleaved caspase-3 further confirmed this (Figure 6B), further suggesting MLKL is not involved in NE-induced apoptosis in vivo. Other than cleavage of caspase-3, we also observed enhanced senescence markers, γH2AX, in the WT lung tissues upon PPE I.N. treatment, which was suppressed by Mlkl depletion (Figure 6A). Furthermore, the immunofluorescence staining also showed that Mlkl depletion suppressed the P21 induction by the PPE I.N. administration (Figure 6C). Collectively, our data further indicated that Mlkl KO suppressed the NE contents in the lung tissue, which compromised the NE-induced cell senescence in the PPE-induced emphysema model.

Figure 6.

Mlkl KO suppressed PPE-induced lung tissue senescence in vivo. The 6∼8-weeks old WT and Mlkl KO C57BL/6 mice were treated with PPE via I.N. instillation. Four weeks later, the lung tissues were dissected. (A) The western blot of NE, cleaved Caspase-3 (C Casp3), and γH2AX in the indicated lung tissues. Left, the representative blots; right, the quantification of indicated proteins. (B) The cleaved caspase 3 in the lung tissues from WT and Mlkl KO mice, which received PPE I.N. instillation, was analyzed by immunofluorescence staining. Left, the representative pictures. Scale bar, 25 µm; right, the quantification of cleaved caspase 3. (C) The P21 in the lung tissues from WT and Mlkl KO mice, which received PPE I.N. instillation, was analyzed by immunofluorescence staining. Left, the representative pictures. Scale bar, 25 µm; right, the quantification of P21. (D) A model of action for MLKL function in NE intracellular transportation. Each experiment was repeated at least 3 times. P (t-test), NS, P > .05; *P < .05; ***P < .001.

Discussion

Previous studies have demonstrated that CS exposure triggers RIPK3/MLKL-mediated necroptosis, which damages lung epithelial cells and induces lung inflammation, ultimately contributing to the development of emphysema in mice.^6,7 However, whether the RIPK3/MLKL signal pathway is also involved in NE-induced lung tissue injury remains unclear. In the present study, we found that necroptosis is not triggered by NE in lung epithelial cells, and the observed NE-induced apoptosis occurs independently of the RIPK3/MLKL axis. Instead, our data reveal a non-canonical, cell death-independent role for MLKL in regulating intracellular NE trafficking. Specifically, MLKL facilitates the nuclear translocation of NE, promoting DNA damage and cellular senescence (Figure 6D). To our knowledge, this is the first report to identify a role for MLKL in mediating intracellular transport of NE, independent of its traditional function in necroptosis. These findings reveal a novel mechanism of NE-induced epithelial injury, providing new insights into the regulation of intracellular protease trafficking.

NE can trigger both cell apoptosis and senescence,^12,15 which are involved in the development and progression of COPD. Apoptosis of lung cells, particularly epithelial and endothelial cells, contributes to the tissue destruction and the progression of emphysema.²⁹ On the other hand, senescence can damage epithelial cells, disrupt epithelial repair, and drive chronic inflammation via the senescence-associated secretory phenotype (SASP), potentially contributing to COPD progression.³⁰ In general, apoptosis is a response to overwhelming stress, leading to cell death, while senescence is a consequence of less severe damage and results in a stable cell cycle arrest.³¹ Consistently, our data showed that NE can trigger lung epithelial cell senescence and apoptosis in a dose-dependent manner. Considering that NE is released by the neutrophil in quantum microbursts,³² epithelial cells are more likely to encounter modest NE concentrations, suggesting that NE-induced senescence may play a vital role in COPD progression. Notably, MLKL depletion selectively attenuated low-dose NE–induced senescence by restricting NE nuclear accumulation and reducing DNA damage, whereas it had no effect on apoptosis induced by higher NE doses. This dissociation is mechanistically plausible, as NE-induced apoptosis has been reported to occur through activation of the CD95 death receptor pathway, independent of DNA damage.¹⁵ Beyond regulating epithelial cell fate, MLKL therefore appears to influence the inflammatory consequences of NE exposure. Senescent epithelial cells secrete a broad array of pro-inflammatory mediators that can amplify and perpetuate lung inflammation.³⁰ By suppressing NE-induced senescence, MLKL deficiency is likely to reduce SASP production, providing a mechanistic explanation for the diminished neutrophil number in BAL observed in MLKL-deficient mice following PPE exposure. Collectively, these findings identify MLKL as a previously unrecognized regulator of NE-driven epithelial senescence and link intracellular NE trafficking to chronic lung inflammation in COPD.

Previous studies have shown that NE transportation can occur via endocytosis, specifically clathrin-mediated endocytosis.²⁷ Following entry into EEA-1⁺early endosomes, NE either traffics to LAMP-1⁺lysosomes for degradation, or “leaks” into the cytosol,²⁷ where it can target intracellular substrates, such as IRS1.²⁶ However, the mechanism by which NE escapes the endosomal compartment is not yet known, and the underlying mechanism that determines the transportation of endosomal NE remains unclear. In this study, we revealed that MLKL plays a role in determining the fate of endosomal NE. Our data showed that MLKL accumulation in the endosome promotes NE release into the cytosol, as depletion of MLKL enhanced the endosomal NE content (Figure 4B), and suppressed the degradation of IRS1, which is a cytosolic target of NE.²⁶ The delivery of endocytosed cargos to lysosomes occurs through direct fusion of late endosomes and lysosomes, which leads to degradation of various cellular components, including NE.²⁷ Our data showed that the lysosome in MLKL KO cells has a higher NE content compared with that of WT cells upon NE treatment (Figure 4D). Inhibition of lysosomal function enhanced the intracellular NE level (Figure 4E). These data suggest that MLKL absence prevents the leakage of NE from the endosome, which ensures that the endosomal NE is delivered to the lysosome for degradation. Consistently, previous studies have indicated that phosphorylated MLKL (activated MLKL) interacts with the lysosomal membrane, leading to the release of lysosomal contents and ultimately promoting necroptosis.³³ However, activated MLKL can be removed from lysosomal membranes through lysosomal degradation, which protects the cell from necroptosis.³⁴ This can explain why NE can’t escape from lysosome degradation after the endosome NE fuses with the lysosome, as the lysosome might degrade MLKL to prevent lysosome contents release. Collectively, our data revealed a novel mechanism for the intracellular transport of NE.

MLKL is a well-known core component of the necrosome that executes necroptotic cell death upon activation by RIPK3.^8,9 However, several studies have revealed that MLKL also contributes to endosome trafficking and the generation of extracellular vesicles (EVs), a process that is not always dependent on RIPK3.^34,35 In addition to translocation to the plasma membrane for necroptosis execution, the activated MLKL may also translocate to subcellular compartments, including mitochondria, exosomes, endosomes, and lysosomes, where it exerts roles in aerobic respiration, leading to the formation of reactive oxygen species, EV biogenesis, cytokine production, endocytosis, and autophagy.^33,34 Moreover, activated MLKL was found to bind to endosomal sorting complexes required for transport (ESCRT), especially ESCRT-III.³⁶ These observations raise the possibility that MLKL may function in the endo-lysosomal system, such as processes along the endocytic and/or autophagic pathways, rather than being restricted solely to necroptosis. However, the implication of this MLKL function in the disease model remains unclear. Here, our data revealed that endosomal MLKL enables NE leakage from the endosome, allowing it to enter the cytosol and nucleus, where it induces DNA damage and senescence, contributing to COPD development. Moreover, MLKL’s ability to undergo nuclei-cytoplasmic shuttling³⁷ raises the possibility that it directly influences NE nuclear localization, further amplifying its impact on epithelial cell fate. These observations broaden the functional landscape of MLKL and highlight its potential role in orchestrating protease trafficking during inflammatory stress. Future investigations should focus on elucidating the molecular determinants of MLKL recruitment to endosomes following NE exposure and clarifying whether MLKL actively mediates NE nuclear translocation. Understanding these pathways significantly broadens our knowledge of MLKL function and NE intracellular transport.

In summary, we have provided evidence that MLKL is involved in NE-induced lung epithelial cell damage by modulating intracellular NE transportation, which is independent of its function in necroptosis. Considering that MLKL-mediated necroptosis contributes to the pathogenesis of CS-induced COPD, MLKL may be a more potent therapeutic target for COPD. While CS is a major cause of COPD, alpha-1 antitrypsin (AAT) deficiency is a significant genetic risk factor that can lead to excess NE activity, contributing to the development of COPD.³⁸ Future studies will focus on investigating the potency of MLKL inhibition or depletion in multiple COPD pre-clinical models, including CS-induced emphysema and spontaneous emphysema caused by AAT mutation.

Author contributions

Dongshi Chen designed and monitored the research. Rong Liu, Aifang Zhang, and Dongshi Chen performed the experiments. Rong Liu and Dongshi Chen analyzed the data and wrote the article. Steven D. Shapiro gave feedback on the draft article.

Conflicts of interest

All authors listed have no financial conflicts of interest related to the submission.

Funding

This work was supported by the U.S. National Heart, Lung, and Blood Institute grant (no. R56HL162749, D.C.), Tobacco-Related Disease Research Program award (no. T35KT9504, D.C.), the American Cancer Society Pilot grant (no. IRG-22-144-60, D.C.), the Wright Foundation (D.C.), and start-up funds from the Keck School of Medicine of USC.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.