Oxidized Phospholipids Aggravate Hepatic Ischemia/Reperfusion Injury by Promoting Macrophage M1 Polarization Via Regulating the Wnt/β-catenin/Autophagy Axis

Abstract

Hepatic ischemia-reperfusion injury (IRI) remains an unavoidable consequence of partial hepatic resection and liver transplantation. Oxidative stress damages cellular lipid membranes, causing the formation of oxidized phospholipids (OxPLs), which are members of damage-associated molecular patterns (DAMPs). Nevertheless, the precise mechanism and significance of OxPLs in hepatic IRI are yet to be investigated. Our findings reveal that OxPLs accumulate excessively in the liver after IR. Compared to the control group, pre-treatment with E06 (an OxPLs-neutralizing antibody) significantly alleviates inflammatory cell infiltration and liver injury following IR. In vitro experiments show that OxPLs inhibit macrophage autophagy, thereby promoting M1 polarization. This regulatory effect depends on the activation of the Wnt/β-Catenin pathway by OxPLs.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02399-x.

Introduction

Hepatic ischemia-reperfusion injury (IRI) is a significant complication of hemorrhagic shock, hepatectomy, and liver transplantation [1–3]. With the global rise in aging populations and metabolic-associated liver disease (MASLD), the liver has become more vulnerable, resulting in more severe IRI [4–6]. Therefore, it is crucial to continue exploring IRI and its impacts.

Macrophages play a central role in orchestrating the inflammatory response during hepatic IRI [7]. As key components of the innate immune system, macrophages are rapidly activated by danger-associated molecular patterns (DAMPs) and hypoxia-induced cellular signals released during tissue injury [8, 9]. Activated macrophages exhibit functional plasticity, polarizing toward either the pro-inflammatory M1 phenotype, which exacerbates tissue damage by producing cytokines [10, 11] like TNF-α and IL-1β, or the anti-inflammatory M2 phenotype, which promotes tissue repair and resolution of inflammation [12, 13]. In hepatic IRI, M1 macrophages dominate in the early phases, contributing to a pro-inflammatory microenvironment that aggravates hepatocyte apoptosis, hepatic sinusoidal endothelial damage, and microcirculatory dysfunction [1, 14]. Conversely, the delayed polarization of M2 macrophages limits the reparative response, prolonging injury.

Phospholipids are essential components of cellular membranes and are abundant throughout the body. Under conditions of oxidative stress—such as tissue injury or inflammation—phospholipids are readily oxidized, giving rise to a diverse group of biologically active products collectively known as oxidized phospholipids (OxPLs) [15, 16]. These molecules have emerged as a subclass of damage-associated molecular patterns (DAMPs) [17, 18], which are generated endogenously in response to sterile or pathogen-induced injury and function as key triggers of innate immune activation [17, 19]. In the context of hepatic ischemia-reperfusion injury (IRI)—a dynamic process involving both ischemic damage and inflammation-driven reperfusion injury—OxPLs are produced primarily during the reperfusion phase. The abrupt reoxygenation leads to a burst of reactive oxygen species (ROS), which attack cellular lipid membranes and promote OxPL formation [16]. Based on current evidence and our immunohistochemical findings, OxPLs in liver IRI are likely generated locally by damaged hepatocytes and other resident liver cells, rather than being derived systemically. One study demonstrated that OxPLs regulate dendritic cell (DC) differentiation and immune responses [20]. In a study on hyperlipidemic mice, researchers cloned the IgM natural antibody E06, which binds to the phosphocholine (PC) headgroup of OxPLs, thereby inhibiting their pro-inflammatory effects and reducing macrophage M1 polarization [21]. In cases of acute injury, OxPLs have been found to induce pulmonary macrophages to cause lung injury and cytokine production [22]. Together, these findings suggest that OxPLs play a context-dependent role as immunomodulatory lipids, particularly in the setting of oxidative stress and tissue injury. Their contribution to the amplification of inflammatory cascades makes them highly relevant in the pathogenesis of hepatic IRI [7].

We report the accumulation of oxidized phospholipids (OxPLs) in the liver and serum of both mouse liver ischemia-reperfusion (IR) models and human subjects. Neutralization of OxPLs through the administration of E06 improved several aspects of hepatic IRI, including the recruitment of inflammatory cells and the reprogramming of macrophages to a pro-inflammatory phenotype. In vivo experiments demonstrated that the accumulation of OxPLs exacerbates liver injury, along with increased recruitment of inflammatory cells to the damaged area. Analysis of the PRJNA438959 dataset revealed that macrophages exposed to higher levels of OxPLs tended to exhibit an M1-like phenotype. Gene ontology (GO) and KEGG enrichment analyses further indicated that genes were enriched in autophagy-related pathways. Extending our investigation, we found that OxPLs promote the inflammatory phenotype of macrophages through the Wnt/β-Catenin/Autophagy axis. Overall, our study provides causal insights into the role of OxPLs in the mechanism of hepatic ischemia-reperfusion injury, and targeting OxPLs may offer an effective therapeutic strategy for improving liver IRI.

Methods

Validation of Clinical Samples

Liver biopsy samples were collected from 44 patients with benign liver diseases who underwent partial hepatectomy combined with the Pringle maneuver. Preoperative and postoperative peripheral blood samples were analyzed for serum levels of alanine aminotransferase (sALT), oxidized phospholipids (OxPLs), and aspartate aminotransferase (sAST). Hepatic biopsies were taken following reperfusion, prior to abdominal closure. The ischemic duration ranged from 12 to 43 min. All participants provided written informed consent, and the study was approved by the ethics committee of Nanjing Medical University.

Mouse Hepatic Ischemia-Reperfusion Model

Hepatic ischemia-reperfusion injury: Partial hepatic warm ischemia in 6–8 weeks old male C57BL/6 mice: Briefly, under isoflurane anesthesia, mice were shaved and disinfected along the A midline laparotomy was performed to expose the liver. After injecting mice with heparin (100 U/kg), mice that underwent IR were subjected to a nonlethal model of segmental (70%) hepatic warm ischemia (90 min) and reperfusion 15 min. Sham mice received the same procedure without using ophthalmic clips to occlude the vessel. Mice were euthanized at various experimental endpoints after reperfusion to obtain liver and serum samples.

For the study of how neutralizing oxidized phospholipids regulates liver injury changes caused by ischemia-reperfusion, the monoclonal antibody E06 of OxPLs (200ug/kg, 330001 S, Avanti), OxPAPC(2 mg/kg, 870604P, Avanti) were injected via the portal vein (P.V.) before closing the abdominal cavity.

Isolation of Primary Hepatocytes

Hepatocytes: In situ collagenase perfusion isolated Murine primary hepatocytes from sham or IR livers. Briefly, murine livers were perfused via the portal vein with HBSS followed by 0.27% collagenase IV (Sigma, St Louis, MO, USA). Isolated perfused livers were dissected and teased through 70 μm nylon mesh cell strainers (BD Biosciences, San Diego, CA, USA) in vitro. Hepatocytes were harvested through centrifuging at 50 g for 3 min/three times.

Extraction of Bone Marrow-Derived Macrophages (BMDMs)

Bone marrow cells were isolated from the femur and tibia of mice. The cells were filtered through a 70 μm mesh, and red blood cells were removed using a red blood cell lysis solution. The cells were cultured in DMEM with 10% FBS and 20% L929-conditioned medium. After 7 days, the culture medium was replaced for subsequent experiments.

Apoptosis Detection

The apoptosis of treated AML12 cells was analyzed by flow cytometry using AnnexinV(APC)—PI(PE) Apoptosis Detection Kit (Biolegend, San Diego California, USA). The treated cells to be analyzed were mixed with AnnexinV-PI and detected by CytoFLEX S (Beckman), and all data were analyzed by FlowJo (V10.8. 1) and CytoExpert (V2.4.0.28).

TUNEL Assay

According to the manufacturer’s protocols, TUNEL staining was conducted using a Tunel kit and freshly frozen tissues. (Servicebio, China) The apoptotic cells were determined and quantified by counting the positive cells in 4 fields with at least 300 cells per field in each group.

In Vitro Hypoxia/Reoxygenation Model

For the hypoxia-reoxygenation(H/R) model, hepatocytes were first cultured in replaced Opti-MEM (GIBCO), and then they were transferred into a hypoxic incubator (5% CO₂, 1% O₂, and 37 °C) for another 12 h. After the hypoxia treatment, the hepatocytes were moved into a normoxic incubator with fresh DMEM containing 10% FBS, and cultured for 0–24 h as previously described.

Detection of OxPLs

The levels of plasma OxPLs in human patients were detected according to the previously described method [23]. For IHC detection of OxPLs in mouse liver sections, OxPLs in the liver were detected with E06-biotin antibody according to the manufacturer’s recommendations (330002 S, Avanti). In short, after fixation in 4% paraformaldehyde and 5% sucrose, the harvested liver was embedded in paraffin. 4 μm thick sections were incubated with E06-Biotin antibody and then developed with a streptavidin-alkaline phosphatase method.

Enzyme-linked Immunosorbent Assay

Mouse serum was collected, and serum TNFα, IL-6, IL-1β, MCP1 and iNOS levels were measured using an ELISA kit (Invitrogen, EPX370-40045-901). The competitive ELISA using E06-IgM was established to measure the concentration of total immunodetectable OxPLs in mouse and human subject serum [23].

Hepatocellular Function Assay

The serum alanine aminotransferase and aspartate aminotransferase levels were measured using the appropriate kits (Servicebio, GM1102, GM1103) to derive the results after an automated biochemical analyzer (Rayto Life and Analytical Sciences Co Ltd Chemray 240).

qRT-PCR

Total RNA was extracted from tissues or cells using Trizol reagent and then reverse transcribed into cDNA. SYBR Green fluorescent dye was used for real-time PCR, and all expression levels and results of the gene of interest were normalized to β-actin expression. Primers used for detection are provided in Supplementary Table 1.

Histology, Immunohistochemistry, and Immunofluorescence Staining

The specimens were fixed in 4% neutral buffered formalin and then embedded in paraffin. Four µm-thick Liver sections were stained with hematoxylin and eosin. The severity of liver IR was graded using Suzuki’s criteria on a scale from 0 to 4 or with antibodies using standard immunohistochemistry protocols. For Immunofluorescence, the fixed tissue sections were placed in a repair kit (Servicebio, China) filled with EDTA antigen retrieval buffer (PH = 9.0) and then boiled in a microwave for repair. After natural cooling, the slides were washed three times with PBS. BSA (3%) in PBS solution was added for 30 min to block nonspecific binding. Then the corresponding primary antibodies were added: anti-CD11b (ab133357, Abcam), anti-S100A9 (ab242945, Abcam), anti-LY6G (ab238132, Abcam), anti-F4/80 (ab6640, Abcam), anti-iNOS (ab283655, Abcam), anti-β-Catenin (ab32572, Abcam). After overnight incubation, the secondary antibody against the corresponding species was added for 50 min in the dark. DAPI was used for the nuclear counterstaining. Slides were observed under a confocal fluorescence microscope (NIKON ECLIPSE C1).

Western Blot

Proteins from liver tissue or cells were lysed with ice-cold RIPA lysis buffer for extraction. Extracted proteins were transferred to PVDF nitrocellulose membranes after running on 10% or 12% SDS-PAGE. ImageJ software was used to quantify western blot bands. Primary antibodies used in the study were provided in Supplementary Table 2.

Statistical Analysis

All repeatable data are presented as mean ± SD. Statistical significance was analyzed using the Student’s unpaired t-test or one-way ANOVA followed by Bonferroni’s method for post hoc pair-wise multiple comparisons. Two-tailed P values less than 0.05 were considered statistically significant.

Results

R1. OxPLs Accumulate in the Liver and Serum in Response To Hepatic ischemia-reperfusion Injury

Previous studies have shown that OxPLs accumulate in the livers of patients with NASH and contribute to inflammation and tissue damage [23]. Based on these findings, we hypothesized that OxPLs might play a similar role in acute liver injury caused by ischemia-reperfusion. We collected 44 surgical specimens from patients undergoing partial hepatectomy (Fig. 1A). Immunohistochemistry with an anti- Oxidized phospholipids antibody (E06) revealed a significant increase in OxPLs in livers exposed to ischemia-reperfusion injury (Fig. 1B-C). Additionally, we observed a positive correlation between serum OxPLs levels and ischemia time, with higher OxPLs levels observed in patients with prolonged ischemia (Fig. 1D). To further investigate this, we constructed a warm ischemia model in mice by occluding the blood supply to the largest hepatic lobe for 90 min, followed by reperfusion at 0, 2, 6, 12, and 24 h. Serum OxPLs levels significantly increased following ischemia-reperfusion, peaking at 6 h, which correlated with the peak of inflammatory damage (Fig. 1E), consistent with previous reports on the inflammatory phase of liver ischemia-reperfusion injury [1, 24]. Next, we used primary hepatocytes in a hypoxia/reoxygenation model (12 h) to simulate ischemia-reperfusion injury. The elevation of OxPLs in the culture supernatants mirrored the in vivo findings, further supporting the role of OxPLs in liver injury (Fig. 1G). Finally, we administered E06, an anti-OxPLs antibody, via the portal vein prior to ischemia-reperfusion. Immunohistochemical analysis showed a reduction in OxPLs accumulation in the liver after E06 treatment, suggesting that neutralizing OxPLs may alleviate their accumulation (Fig. 1H-I). In conclusion, our data show that OxPLs accumulation in the liver and serum is associated with ischemia-reperfusion injury and may contribute to more severe inflammatory damage.

Fig. 1.

OxPLs accumulate in the liver and serum in response to hepatic ischemia-reperfusion injury. A. Schematic diagram showing the detection of OxPLs content in liver and serum samples by immunohistochemistry and ELISA (n = 44); B. OxPLs expression was detected by IHC in liver samples of patients before and after hepatic portal blockade (scale bar: 100 μm); C. ELISA detected OxPLs content in the serum of patients before and after hepatic portal blockade; D. Correlation analysis between the ischemia time during partial hepatectomy and the difference in OxPLs content in serum before and after the blockade; E. A warm ischemia model of mouse liver was constructed, with E06 (200 µg/kg, 330001 S, Avanti) or PBS injected via the portal vein (P.V.), followed by 6 h of reperfusion before harvesting mouse liver tissues (n = 5); F. Detection of OxPLs content in mouse serum (n = 5); G. After 12 h of H/R, the culture supernatant of primary hepatocytes was collected to detect OxPLs content (n = 5); H. Immunostaining of the liver of mice after 6 h of IR using biotinylated E06 IgM (scale bar: 20 μm); I. Quantification of hepatic OxPLs immunohistochemistry (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

R2. Neutralization of OxPLs Ameliorates Hepatic Ischemia-Reperfusion Injury

Next, we investigated whether the levels of OxPLs in the liver and serum were associated with liver injury. Upon examining the clinical samples, we found that serum OxPLs levels were positively correlated with the degree of liver injury (Fig. 2A-B). H&E staining revealed that hepatic ischemia-reperfusion injury was significantly alleviated in the E06-treated mice (Fig. 2G-H), and tissue damage, as assessed by the Suzuki score, was notably lower in the E06 group (Fig. 2L). Furthermore, the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells was significantly reduced in the E06 group compared to the PBS group (Fig. 2G-I). Serum alanine aminotransferase (ALT) levels were also lower in the E06-treated mice (Fig. 2J-K). We isolated hepatocytes from the livers of mice that underwent 6 h of reperfusion using differential centrifugation. Flow cytometry and Western blot analysis were performed to assess hepatocyte apoptosis, and we observed a trend toward a reduction in total apoptosis in the E06 group (Fig. 2C-D), suggesting that OxPLs contribute to the exacerbation of liver damage. Interestingly, administration of OxPLs further exacerbated liver injury following ischemia-reperfusion. By injecting OxPLs through the portal vein, combined with H&E and TUNEL staining, we observed significantly more severe liver ischemia-reperfusion injury in the OxPLs-treated mice (Fig. S2A-C), with elevated serum transaminase levels (Fig. S2D-E). Furthermore, we found that in mice treated with OxPLs, hepatocytes experienced more severe mitochondrial membrane potential collapse (Fig. S1A, C), which indicates more pronounced cellular apoptosis.

Fig. 2.

Neutralization of OxPLs ameliorates hepatic ischemia-reperfusion injury. A, B. Correlation analysis between sALT, sAST, and the difference in OxPLs content in serum before and after hepatic portal blockade during partial hepatectomy (n = 44); C. Primary hepatocytes were extracted from mouse liver after ischemia-reperfusion, and apoptosis was detected by flow cytometry (n = 3); D. The expression of BAX and BCL-2 was detected by western blot (n = 3); E. Quantification of apoptotic cells (n = 3); F. Quantification of BAX and BCL-2 grayscale values (n = 3); G. Liver tissue damage was observed by HE staining and TUNEL staining (scale bar: 20 μm, 100 μm) in mouse models (n = 5); H, I. Quantification of IR area and apoptotic cells (n = 5); J, K. Measurement of serum alanine aminotransferase and serum aspartate aminotransferase levels in the livers of WT and KO mice subjected to IR (n = 5); L. Assessment of liver tissue injury induced by IR using Suzuki’s histologic criteria (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

R3. OxPLs Exacerbate the Inflammatory Response Following Liver Ischemia-Reperfusion Injury

Damage-Associated Molecular Patterns (DAMPs) play a crucial role in activating the innate immune response and subsequent inflammatory cell recruitment during liver ischemia-reperfusion injury (IRI) [25, 26]. We sought to determine whether OxPLs could act as DAMPs and contribute to the inflammatory response in liver IRI. To assess this, we examined the expression of CD11b, S100A9, and LY6G in the liver, which reflect the recruitment of neutrophils and monocytes to the liver injury site [27]. In the IRI mice, the expression of these representative inflammatory cell markers was significantly elevated. Notably, additional administration of OxPLs further increased the recruitment of inflammatory cells, while treatment with E06, an anti-OxPLs antibody, reversed this effect (Fig. 3A). We also measured the expression of chemokines in the liver and found that CXCL1, CXCL2, and CXCL5 were upregulated (Fig. 3B-D). In addition, serum levels of the inflammatory cytokines interleukin (IL) 1β, tumor necrosis factor alpha (TNFα), and monocyte chemotactic protein-1 (MCP1) were significantly higher in the IR6h + OxPLs group compared to the IR6h group (Fig. 3E, G). Using flow cytometry, we further validated the liver inflammatory factors (IL-1β, IL-6, IL-17 A, IL-23, MCP-1, IFN-β, IFN-γ, GM-CSF, IL-12p70, TNF-α, IL-10). The expression of these inflammatory cytokines was elevated in the IRI mouse model, confirming the successful establishment of the liver IRI model. Notably, the additional administration of OxPLs further promoted inflammation, while E06 treatment alleviated liver inflammation (Fig. 3F).

Fig. 3.

OxPLs exacerbate the inflammatory response following liver ischemia-reperfusion injury. A. Tissue of CD11b, S100A9, and LY6G protein immunofluorescence for E06+/-, OxPL+/- IR6h liver neutrophil infiltration (scale bar: 50 μm) (n = 5); B-D. Chemokines(CXCL1,CXCL2, CXCL5) mRNA expression levels were analyzed (n = 5); E,F. Postoperative serum levels of cytokines (TNFα,IL-6,IL-1β,and MCP1) in mice were measured by enzyme-linked immunosorbent assay (n = 5); G. Inflammatory cytokines in livers of those subjected to IRI and control group. (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

R4. OxPLs Promote M1 Macrophage Polarization in Liver during Hepatic Ischemia-Reperfusion Injury

Macrophages can be classified into pro-inflammatory M1 and anti-inflammatory M2 phenotypes based on their surface markers and cytokine release profiles. In liver ischemia-reperfusion injury (IRI), excessive activation of the pro-inflammatory M1 phenotype has been shown to exacerbate tissue damage and inflammation [11, 28]. To explore whether OxPLs play a role in macrophage polarization during liver IRI, we investigated the impact of OxPLs on macrophage M1 polarization. We performed tissue immunofluorescence staining for F4/80 and iNOS to identify M1 macrophages in the liver. Our results revealed that M1 polarization of macrophages was significantly reduced in the E06-treated group (Fig. 4A, E). Furthermore, compared to the IR6h group, the IR6h + OxPLs group exhibited higher levels of M1 markers, iNOS and CD86, while the IR6h + E06 group showed the opposite pattern (Fig. 4B-C). These findings were further corroborated by immunohistochemistry (Fig. 4D, G). Next, we extracted single-cell suspensions from the ischemic reperfused livers of mice and analyzed them using flow cytometry. We found that the proportion of F4/80 + CD86 + macrophages was highest in the IR6h + OxPLs group (Fig. 4F, H), which is consistent with our previous findings. To determine whether hepatic macrophages are responsible for OxPL-mediated liver injury, clodronate liposomes were used to deplete macrophages [29]. As shown by F4/80 immunofluorescence staining, nearly all hepatic macrophages were eliminated in the clodronate-treated group compared with the control liposome group (Fig. S4A). Macrophage depletion not only reversed liver injury induced by hepatic I/R but also mitigated the exacerbated damage that would otherwise occur upon additional OxPL administration (Fig. S4A-D). Interestingly, co-administration of E06 and clodronate liposomes did not produce an additive effect, suggesting that macrophages are indispensable for transducing OxPL-induced inflammatory signals. In summary, our study demonstrates that OxPLs accumulation leads to excessive activation of macrophages, particularly through the promotion of M1 polarization. This excessive activation of macrophages likely contributes to the exacerbation of inflammation, which may be a critical factor in the progression of liver ischemia-reperfusion injury.

Fig. 4.

OxPLs Promote M1 Macrophage Polarization in Liver during Hepatic Ischemia-Reperfusion Injury. A. F4/80, iNOS stained liver tissue representative immunofluorescence images (scale bar:100 μm) (n = 5); B, C. The expression of NOS2 and CD86 in the liver after IR was detected by qPCR (n = 4); D. Immunohistochemistry staining of iNOS in 6 h IR liver tissues or sham liver tissues (scale bar: 100 μm) (n = 5); E. Quantification of the percentage of M1-like macrophages by flow cytometry (n = 5); F. Flow cytometry analysis of hepatic macrophages isolated from the livers of mice (n = 5); G. Quantification of hepatic iNOS immunohistochemistry (n = 5); H. Quantification of the percentage of M1-like macrophages by flow cytometry (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

R5. OxPLs Regulates Macrophage M1 Polarization in Vitro

To investigate the effect of OxPLs on macrophage polarization, in vitro studies were conducted using RAW264.7 cells.The viability of RAW264.7 cells treated with different concentrations of OxPLs (0–50 µg/mL) was assessed using the CCK8 assay. The results showed that OxPLs had no significant effect on cell viability within the concentration range of 1–10 µg/mL. However, when the concentration of OxPLs exceeded 20 µg/mL, cell viability was significantly inhibited (survival rate < 80%, Fig. 5A). Therefore, 5, 10, and 15 µg/mL concentrations of OxPLs were selected for subsequent experiments.To study the impact of OxPLs on macrophage polarization, RAW264.7 cells were induced to undergo M1 polarization using LPS and IFNγ, followed by OxPLs treatment. Flow cytometry was used to assess CD86 expression, while western blot (WB) analysis was used to detect the co-expression of CD80/iNOS. Additionally, RT-qPCR was employed to determine the relative expression levels of M1-associated markers and pro-inflammatory cytokines, such as CD80, CD86, TNF-α, IL-1β, and NOS2. Flow cytometry results revealed that after LPS and IFNγ stimulation, CD86 expression was significantly upregulated, and OxPLs treatment further increased this expression (Fig. 5B-C). WB results indicated that OxPLs treatment further enhanced the co-expression of CD80 and iNOS (Fig. 5D-F). Additionally, the mRNA levels of CD80 and CD86 showed a similar trend (Fig. 5G-H). RT-qPCR results confirmed the successful establishment of the M1 polarization model, with LPS and IFNγ inducing significant upregulation of TNF-α, IL-1β, and NOS2, which was further upregulated under the stimulation of OxPLs (Fig. 5I-K). Furthermore, ELISA analysis of the culture supernatants revealed that OxPLs treatment significantly increased the secretion of TNF-α, IL-1β, and iNOS in LPS/IFNγ-induced RAW264.7 cells (Fig. 5L-N). These results suggest that OxPLs promotes M1 polarization in macrophages.

Fig. 5.

OxPLs regulates macrophage M1 polarization in vitro. A. The cell viability of RAW264.7 cells after treatment with different concentrations of OxPL for 2 h, 4–6 h was detected by CCK8 method (n = 5); B, C. The expression of CD86 in RAW264.7 cells after treatment with LPS/IFNγ and OxPLs for 4 h was detected by flow cytometry, and the percentage of CD86 + RAW264.7 cells after treatment with LPS/IFNγ and OxPLs for 4 h was obtained by flow cytometry (n = 3); D. The expression of CD80 and iNOS was detected by western blot (n = 3); E, F. Quantification of CD80 and iNOS grayscale values (n = 3); G-K. The expression of CD80, CD86, TNFα, IL-1β, and NOS2 in RAW264.7 cells after treatment with LPS/IFNγ and OxPLs for 4 h was detected by qPCR (n = 3); L-N. The contents of TNF-α, IL-1β and iNOS in culture supernatants of RAW264.7 cells after treatment with LPS/IFNγ and OxPLs for 4 h were detected by ELISA (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

R6. OxPLs Promotes Macrophage Pro-Inflammatory Phenotype Reprogramming by Impairing Autophagy

We further investigated how OxPLs regulates macrophage phenotypic changes. Analysis of the transcriptomic data of E06+/- macrophages from the National Center for Biotechnology Information Sequence Read Archive (PRJNA438959) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that OxPLs might promote macrophage phenotypic changes by affecting autophagy-related functions (Fig. 6A-B). Western blot (WB) was used to assess the expression of autophagy-related proteins such as Beclin1, P62, and LC3. We found that adding OxPLs to the macrophage culture medium resulted in significant M1 polarization, consistent with our previous findings. However, there was no significant change in the expression of autophagy-related proteins, possibly because autophagy was not activated. Therefore, we added OxPLs in the presence of Rapamycin, and this led to noticeable changes in the expression of autophagy-related proteins. After OxPLs stimulation, the expression of LC3-II and Beclin1 decreased, while P62 expression increased (Fig. 6C-G). We then used monodansylcadaverine (MDC) as a probe to specifically label autophagosomes. After OxPLs treatment, we observed autophagy inhibition in macrophages (Fig. 6H-I). In conclusion, our study demonstrates that OxPLs promotes M1 phenotypic conversion by impairing macrophage autophagy.

Fig. 6.

OxPLs Promotes Macrophage Pro-Inflammatory Phenotype Reprogramming by Impairing Autophagy. A. Heat map of RNA-seq data from Ldlr−/− and Ldlr−/−/E06-scFv TGEM collected after 16 weeks of HCD, and the genes related to inflammatory response and autophagy are listed. (n = 4); B. KEGG enrichment analysis reveals the major signaling pathways in the TGEM of E06-scFv or WT mice fed HCD, as identified by RNA-seq. The significantly up-regulated pathways are annotated by red dashed boxes; C. The expression of iNOS, Beclin1, P62, and LC3B was detected by western blot (n = 3); D-G. Quantification of iNOS, Beclin1, P62, and LC3B grayscale values (n = 3); H. Autophagosomes were specifically labeled by monodansylcadaverine (MDC) (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

R7. OxPLs-dependent Wnt/β-catenin Pathway Activation Exacerbates Macrophage M1 Polarization Through Autophagy Inhibition

We further investigated how OxPLs regulates macrophage polarization. Analysis of the transcriptomic data from E06+/- macrophages (PRJNA438959) revealed that the Wnt/β-catenin pathway had the most enriched genes (Fig. S3A). Therefore, we explored whether OxPLs modulates macrophage polarization by regulating the Wnt/β-catenin pathway in the context of liver ischemia-reperfusion injury. Since nuclear translocation of β-catenin is a key event in Wnt/β-catenin pathway activation [30], we examined the distribution of β-catenin in the cytoplasm and nucleus. Western blot analysis of OxPLs-treated BMDMs showed an increased nuclear localization of β-catenin (Fig. 7A, D), which was confirmed by immunofluorescence (Fig. 7B).

Fig. 7.

OxPLs-dependent Wnt/β-catenin pathway activation exacerbates macrophage M1 polarization through autophagy inhibition. Cytoplasmic and nuclear proteins in RAW264.7 cells were isolated and Western blot analysis was performed to verify the nuclear translocation of β-catenin. RAW264.7 cells were treated with OxPLs or PBS for 4 h (n = 3). A. Representative Western blot on the relative abundance of cytoplasmic and nuclear β-catenin protein in two groups are presented; B. Immunofluorescence staining demonstrated that the expression and nuclear translocation of β-catenin were induced by OxPLs in RAW264.7 cells (scale bar: 10 μm, 50 μm) (n = 3); RAW264.7 cells were treated with OxPLs, followed by stimulation with ICG-001 (5 µM) for 24 h. C The expression of Beclin1, P62, and LC3B was detected by western blot (n = 3), D. Quantification on the relative abundance of cytoplasmic and nuclear β-catenin protein (n = 3). RAW264.7 cells were treated with OxPLs, followed by treatment with or without ICG-001 (5 µM, 24 h) and RAPA (200 nM, last 2 h) as indicated, E. The expression of Beclin1, P62, and LC3B was detected by western blot (n = 3); F. Quantification of Beclin1, P62, and LC3B grayscale values (n = 3); RAW264.7 cells were treated with OxPLs, followed by treatment with or without ICG-001 (5 µM, 24 h) and 3-MA (5 mM, last 2 h) as indicated, G. The expression of Beclin1, P62, and LC3B was detected by western blot (n = 3); H, I. Quantification of Beclin1, P62, CD80, LC3B and iNOS grayscale values (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Our Western blot results indicated that OxPLs treatment inhibited autophagy in BMDMs, as evidenced by decreased expression of autophagy markers LC3-II and Beclin-1, and an increase in the expression of autophagic cargo protein SQSTM1/p62. However, ICG-001, a specific inhibitor of the Wnt/β-catenin pathway, reversed these effects (Fig. 7C, F). These results suggest that OxPLs inhibits autophagy by activating the Wnt/β-catenin pathway.

Given that OxPLs/Wnt/β-catenin signaling inhibits autophagy, we next examined the role of autophagy in OxPLs/Wnt/β-catenin-induced macrophage M1 polarization. We first used rapamycin (Rapa) as an autophagy inducer. Rapa treatment led to an increase in LC3-II and Beclin-1 expression and a decrease in SQSTM1 levels (Fig. 7E, H), confirming successful autophagy activation. Notably, Rapa inhibited OxPLs-induced macrophage M1 polarization to a similar degree as ICG-001, as shown by reduced expression of iNOS and CD80 (Fig. 7E, H). In contrast, treatment with 3-Methyladenine (3-MA), a late-stage autophagic flux inhibitor, induced macrophage M1 polarization, as evidenced by elevated levels of iNOS and CD80, similar to the effects of OxPLs. Additionally, 3-MA reversed the inhibitory effect of ICG-001 on OxPLs-induced M1 polarization (Fig. 7G-I). In conclusion, our findings suggest that the inhibition of autophagy by OxPLs/Wnt/β-catenin signaling is crucial for promoting macrophage M1 polarization.

Discussion

Liver ischemia-reperfusion injury (IRI) is a significant cause of liver dysfunction, particularly following liver transplantation and surgeries. Understanding the underlying mechanisms of IRI is essential for improving patient outcomes. This study focuses on the role of oxidized phospholipids (OxPLs) in liver IRI, particularly given the increasing prevalence of metabolic-associated liver diseases (MASLD) and an aging population, which heightens liver vulnerability to ischemia-reperfusion injury [31, 32]. Our findings show that the accumulation of OxPLs is closely linked to liver damage during IRI. Neutralizing OxPLs using the E06 antibody reduced their accumulation in the liver, alleviating liver injury, thus confirming the critical role of OxPLs in IRI-associated inflammation.

Macrophages play dual roles in liver IRI, with M1-like polarization initiating and exacerbating inflammation through the secretion of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 [33, 34]. In this study, we found that OxPLs significantly promoted M1 polarization in macrophages, as evidenced by increased expression of M1 markers like CD86 and iNOS. This supports the notion that OxPLs, acting as damage-associated molecular patterns (DAMPs), play a pivotal role in activating innate immune responses in IRI. These findings emphasize the potential of targeting macrophage polarization as a strategy to mitigate IRI-induced liver damage.

Autophagy, a cellular process crucial for maintaining homeostasis, regulates macrophage polarization and inflammation [35, 36]. Recent studies have shown that autophagy influences immune responses by modulating macrophage polarization [37, 38]. Inhibition of autophagy promotes M1 polarization, which enhances inflammation and tissue damage, whereas autophagy activation can limit excessive M1 polarization and promote M2 polarization, reducing inflammation and aiding tissue repair [39, 40]. In this study, we demonstrated that OxPLs inhibit autophagy in macrophages by activating the Wnt/β-catenin pathway. This autophagy inhibition leads to persistent M1 polarization, which exacerbates inflammation and liver injury, suggesting that autophagy plays a protective role by regulating macrophage polarization and limiting excessive inflammation. Dysregulation of this process, induced by OxPLs, contributes to the progression of liver IRI.

The implications of our study extend beyond liver IRI and could have relevance to other liver diseases marked by chronic inflammation, such as MASLD. Since DAMPs, including OxPLs, are involved in various inflammatory conditions, targeting OxPLs and their signaling pathways could provide therapeutic benefits for a broader range of liver diseases.

However, there are limitations to our study. The sample size of clinical specimens was relatively small, and our experiments were predominantly conducted using mouse models, which may not fully replicate the complexity of human diseases. Future studies should include larger patient cohorts and explore the role of OxPLs in various liver diseases to validate the generalizability of our results. Additionally, factors like age, gender, and comorbidities affecting OxPL levels should be addressed in future studies. Another limitation is that our study focused on macrophages, while OxPLs may also affect other immune cells, such as Tregs, where oxPAPC has been shown to impair its stability and function. This broader immunomodulatory role warrants further investigation.

In conclusion, our study underscores the pivotal role of OxPLs in liver IRI and their potential as therapeutic targets. By promoting M1 macrophage polarization and inhibiting autophagy, OxPLs exacerbate inflammation and liver damage. These findings pave the way for the development of targeted therapies to mitigate liver IRI and other related liver diseases. Future research should focus on validating these findings in larger and more diverse patient populations, and on translating these preclinical insights into clinical applications.

Conclusions

In conclusion, our study demonstrates that liver ischemia-reperfusion (IR) leads to excessive accumulation of OxPLs. OxPLs induce macrophage M1 polarization by regulating the OxPLs/Wnt/β-catenin/Autophagy axis, which contributes to more severe inflammatory damage in the liver. Therefore, administration of the IgM natural antibody E06, which targets OxPLs, represents a potential strategy for treating postoperative liver injury in patients undergoing liver resection.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

IRI

Ischemia-reperfusion injury

OxPLs

Oxidized phospholipids

sALT

Serum alanine aminotransferase

sAST

Serum aspartate aminotransferase

DAMPs

Damage-associated molecular patterns

MASLD

Metabolic-associated liver disease

ROS

Reactive oxygen species

BMDMs

Bone marrow-derived macrophages

Author Contributions

MHC designed the study. MHC, YL, and JL carried out the experiments; Data analysis was performed by ZYW, YZ, XJC, XDL, NX, WJY, LFS, JW, LYP and SH; Funding Acquisition: LYP. All authors read and approved the final manuscript.

Funding

This project was supported by grants from the National Natural Science Foundation of China (NSFC) (Grant Nos. 81870443 and 81273261).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethical Approval and Consent to Participate

Ethics approval and consent to participate in all the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (IACUC-2404110).

Ethical approvalfor this study was obtained from the Ethics Committee of Nanjing Medical University (2023-SR-390), and written informed consent was obtained from all participants according to the principles outlined in the Declaration of Helsinki.

Consent for Publication

Not applicable.

Competing interests

The authors declare no competing interests.