Olive Oil-Based Lipid Emulsion Ameliorates Immune Checkpoint Inhibitor-Induced Myocarditis via Inhibition of the NF-κB/NLRP3/IL-1β Pathway

Summary

Olive oil-based lipid emulsions mitigate immune checkpoint inhibitor-induced myocarditis by targeting the NF-κB/NLRP3/IL-1β pathway, demonstrating their potential in suppressing inflammatory cascades. These findings highlight its promise as an adjunctive therapy for immune-related cardiac injury.

Abstract

The present study aimed to investigate the therapeutic efficacy and underlying mechanism of olive oil-based lipid emulsions (OOLE) in mitigating immune checkpoint inhibitor (ICI)-induced myocarditis triggered by ipilimumab (IPI) and nivolumab (NIVO). An in vitro model of inflammatory cardiomyocytes was established by co-culturing HL-1 cells with CD4⁺/CD8⁺ T cells isolated from ICI-treated mice. Cells were treated with 10% OOLE, followed by flow cytometry for apoptosis, ELISA for cytokine profiling (TNF-α, IL-1β, IL-6), and Western blot/qPCR for pathway analysis (NF-κB, NLRP3, IL-1β). In vivo, myocarditis was induced in mice via IPI/NIVO administration. Cardiac function was assessed using echocardiography, and inflammatory markers were evaluated in serum and myocardial tissue. The results showed that OOLE significantly reduced T cell-induced apoptosis and suppressed inflammatory cytokine production in HL-1 cells, while the expression of NF-κB, NLRP3, and IL-1β was downregulated. In vivo., OOLE improved left ventricular functional parameters and attenuated systemic inflammation. Molecular analyses confirmed that these protective effects were mediated via the inhibition of the NF-κB/NLRP3/IL-1β signaling axis. In conclusion, OOLE mitigates acute ICI-induced myocarditis by targeting the NF-κB/NLRP3/IL-1β pathway, demonstrating its potential in suppressing early inflammatory cascades and providing rapid cardioprotection. These findings highlight its promise as an adjunctive therapy for immune-related cardiac injury.

Introduction

Immune checkpoint inhibitors (ICIs), such as those targeting programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), have revolutionized the treatment landscape for various malignancies. However, the widespread clinical application of ICIs is frequently complicated by immune-related adverse events (irAEs). Among these, ICI-induced myocarditis is a rare but potentially fatal complication, characterized by a rapid clinical course and high mortality rates ranging from 25% to 50%^1,2. Despite its severity, current therapeutic options for ICI-induced myocarditis remain largely limited to high-dose corticosteroids and non-specific immunosuppressants^3,4. These traditional interventions often yield suboptimal responses and may compromise the anti-tumor efficacy of ICIs¹, creating a critical knowledge gap in the search for safer and more targeted cardioprotective strategies that can mitigate cardiac inflammation without systemic immunosuppression.

The NOD-like receptor protein 3 (NLRP3) inflammasome, activated via the NF-κB signaling pathway, has emerged as a central driver of the cytokine storm observed in ICI-induced myocarditis^5,6. Excessive production of pro-inflammatory cytokines, particularly IL-1β and IL-6, leads to profound cardiomyocyte injury and heart failure⁷. Although several lipid-based interventions have been explored in cardiovascular diseases, the potential of olive oil-based lipid emulsions (OOLE) in this specific context remains unexplored. OOLE is rich in oleic acid, a monounsaturated fatty acid known for its unique ability to modulate membrane fluidity and suppress inflammatory cascades^8,9. However, whether OOLE can specifically ameliorate the hyper-inflammatory state characteristic of ICI-induced myocarditis has not been established.

In this study, we aim to address this gap by investigating the therapeutic potential of OOLE. We hypothesize that OOLE exerts a potent cardioprotective effect by inhibiting the NF-κB/NLRP3/IL-1β signaling axis, thereby suppressing the inflammatory surge and preserving cardiac function. By combining an in vitro co-culture model of HL-1 cardiomyocytes and T cells with an in vivo. mouse model of ICI-induced myocarditis, we demonstrate that OOLE intervention provides rapid functional recovery and mitigates myocardial injury. This study provides the first evidence for OOLE as a novel, targeted nutritional pharmacological approach to managing ICI-related cardiotoxicity.

Protocol

All procedures were approved by the Animal Ethics Committee of Guangzhou Miles Biotechnology Co., Ltd., with approval number IACUC-MIS2023076. The company mentioned is an animal experimentation center; our animal experiments are conducted here and are therefore subject to review by the center's ethics committee.

Animals and experimental design
Thirty BALB/c mice (male, 8 weeks old, weighing 22 ± 2 g) were purchased commercially. Model induction and validation criteria: An immune checkpoint inhibitor (ICI)-induced myocarditis model was established by intravenous injection of ipilimumab (5 mg/kg) and nivolumab (10 mg/kg) for 2 consecutive weeks. Dosing occurred every 3 days for a total modeling duration of 2 weeks. This protocol has previously been shown to induce significant immune-mediated cardiac injury¹⁰. At 24 h after the final injection, blood samples were collected via the retro-orbital plexus and centrifuged at 300 x g. for 15 min at 4 °C to isolate the serum; subsequently, serum cTnT levels were quantified using a specific ELISA kit, involving sequential steps of sample loading, HRP-conjugated antibody incubation, automated washing, and TMB chromogen reaction, with the optical density measured at 450 nm to calculate concentrations based on a four-parameter logistic curve, and a cTnT concentration ≥10 ng/mL was considered a successful model establishment.

Six model mice with the closest body weight and cTnT concentration were randomly divided into two groups: a control group and a treatment group (n=3 for both, biological replication). The control group received daily intravenous injections of normal saline at a dose of 5 mL/kg. The treatment group received daily intravenous injections of 5% olive oil-based lipid emulsion (OOLE) at a dose of 5 mL/kg via the tail vein for 6 days (this represents the dosage we employ for clinical treatment within our institution; it was extrapolated for use in mice based on body surface area ratios). The following tests were performed on a 6-day observation window: Cardiac function was assessed at baseline (day 0) and post-treatment (day 6) using an ultrasound photoacoustic imaging system. Blood was collected via the retroorbital vein on day 6, and serum cytokines were quantified by ELISA. After euthanizing the animals with an overdose of anesthesia, the heart tissue was flash-frozen for protein analysis.

T cell isolation
The mice in the control group were euthanized with an overdose of anesthesia. The spleens were aseptically excised after a 5 min surface sterilization in 75% ethanol, and all connective tissues were removed. The spleens were mechanically dissociated using a 70 µm cell strainer with PBS supplemented with 2% FBS to create a single-cell suspension. This suspension was then centrifuged at 300 x g., 4 °C for 5 min, and erythrocytes were lysed with 1 mL of ACK Lysing Buffer for 2 min at room temperature. The lysis process was terminated by washing with PBS. T cells were negatively enriched to prevent activation using a mouse T cell isolation kit. For every 10 million cells, 50 µL of selection cocktail was added and incubated at room temperature for 10 min. This was followed by the addition of 75 µL of streptavidin magnetic beads per 10 million cells, with a 5 min incubation. Bead-bound cells were retained using a magnet for 3 min, and the enriched T cells (with over 94% purity) were collected from the supernatant. For sorting the CD4⁺ and CD8⁺ subsets, the enriched cells were stained with anti-CD3ε-PE, anti-CD4-APC, and anti-CD8-FITC antibodies for 15 min in the dark, then washed 3x with pre-cooled PBS. Sorting was performed on a flow cytometer using the following gating strategy: lymphocytes (FSC/SSC), then CD3⁺, then CD4⁺ or CD8⁺ subsets. Cells were sorted in Purity Mode into collection tubes pre-filled with 200 µL of PBS, yielding target subsets with greater than 98% purity.

Construction and treatment of the myocarditis cell model
The HL-1 cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) under standard conditions (37 °C, 5% CO₂). Cells were divided into three groups: ​Control group (untreated), model group (HL-1 cells treated with CD4+/CD8+ T cells for 24 h), and OOLE group (Model group co-treated with 10% OOLE for 24 h).

ELISA for serum detection of myocardial injury-related proteins
Incubate 50 µL of serum sample in the pre-coated antibody-rich microplate wells of the kit for 30 minutes, then wash three times with PBST to remove unbound components. Add the prepared horseradish peroxidase (HRP)-labeled avidin solution for a second incubation. Wash three times again with PBST, add 100 µL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate to initiate the colorimetric reaction, and terminate the reaction with 100 µL of stop solution within 30–45 minutes after initiation. Measure the absorbance (OD) at 450 nm using a microplate reader and quantify the concentration of the target analyte using a standard curve plotted with known reference standards.

Western blot
Cardiac tissues were pulverized using a tissue homogenizer with liquid nitrogen. Subsequently, both tissue and cellular samples were lysed in 200 µL of RIPA buffer on ice for 30 min. The lysates were centrifuged at 12,000 x g. for 15 min at 4 °C to collect protein supernatants. Protein concentrations were determined using a BCA protein assay kit. After adjusting the protein concentration to 5 µg/µL, the samples were mixed with 5× loading buffer and denatured by heating at 95 °C for 10 min. Proteins were separated on 10% SDS-PAGE gels (run at 100 V for 90 min) and subsequently transferred to PVDF membranes via semi-dry electroblotting at 15 V for 30 min. The membranes were blocked with 5% non-fat milk and then probed overnight at 4 °C with primary antibodies (including IL-1β, NLRP3, MyD88, NF-κB p65, and Tubulin; 1:1,000 dilution)². After washing the membranes three times for 10 min each with Tris-buffered saline containing 0.1% Tween 20 (TBST), they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 dilution) for 1 h at room temperature. Protein bands were visualized by applying an enhanced chemiluminescence (ECL) substrate, and the signals were captured using a chemiluminescence imaging system. Finally, relative expression levels were quantitatively analyzed by normalizing the densitometric values of the target protein bands to the Tubulin internal control using ImageJ software.

Cell apoptosis assay by flow cytometry
The HL-1 cells were washed 2x with phosphate-buffered saline (PBS). For cell dissociation, 1 mL of trypsin was added and incubated for 1 min, followed immediately by neutralization with 2 mL of complete culture medium. The cell suspension was then centrifuged at 250 x g. for 5 min at 4 °C, and the supernatant was discarded. The collected cell pellet was washed 2x with ice-cold PBS and resuspended in 1x binding buffer to achieve a cell density of 1 x 10⁶ cells/mL. A 100 µL aliquot of this cell suspension was transferred into a flow cytometry tube, followed by the addition of 5 µL of FITC-Annexin V and 5 µL of propidium iodide (PI). After gentle vortexing, the mixture was incubated for 15 min at room temperature (25 °C) in the dark. Finally, 400 µL of 1 x binding buffer was added to each tube, and cell apoptosis was subsequently analyzed using a flow cytometer. For the gating strategy, events were first gated on forward scatter (FSC) and side scatter (SSC) to exclude subcellular debris and isolate the intact HL-1 cell population. Within this primary gate, apoptotic cells were quantified using quadrant analysis, with early apoptotic cells defined as FITC-Annexin V⁺/PI⁻ and late apoptotic cells defined as FITC-Annexin V⁺/PI⁺.

Statistical analysis
Each experiment was performed in triplicate, and statistical analysis was conducted using statistical software. Differences between two groups were analyzed using the independent samples t-test; differences among three or more groups were analyzed using one-way ANOVA followed by the Bonferroni post-hoc test. Data are expressed as mean ± standard error (SEM). p < 0.05 was considered statistically significant.

Results

Ipilimumab and Novolumab combination induce ICI-induced Myocarditis in an animal model
To establish an ICI-induced myocarditis model, BALB/c mice were administered intravenous injections of ipilimumab (5 mg/kg) and nivolumab (10 mg/kg) via the tail vein. Serum levels of cardiac troponin T (cTnT) were subsequently measured using an enzyme-linked immunosorbent assay (ELISA). A cTnT level ≥ 10 ng/mL was defined as the threshold for successful model induction. As shown in Figure 1A

Discussion

In this study, we demonstrated for the first time that olive oil-based lipid emulsions (OOLE) effectively attenuate immune checkpoint inhibitor (ICI)-induced myocarditis by suppressing the IL-1β/NLRP3/NF-κB signaling pathway. Our results provide both mechanistic and functional evidence that OOLE not only improves cardiac function but also alleviates myocardial inflammation in both in vivo and in vitro. models of ICI-induced myocarditis.

Mechanistically, the findings...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22um PVDF membranes	Millipore	ISEQ00010
4% paraformaldehyde	Guangzhou Jadeite Biotechnology Co., Ltd.	BL539A
BALB/c mice	Guangdong Zhiyuan Biomedicine Technology Co., Ltd.
BCA assay kit	Dingguo Biotech	BCA02
bright-field microscopy	MSHOT	ml31
cTnT elisa kit	lunchangshuobiotech	ED-28107
Electrophoresis apparatus	WIX	miniPro4
Fully Automated Chemiluminescence Image Analysis System	FUJIFILM	LAS-3000
hematoxylin-eosin kit	Guangzhou Jadeite Biotechnology Co., Ltd.	BL2236A
IFN-γ ELISA kits	Xiamen Lenchangshuo Biotechnology Co., Ltd. (Fujian, China).	ED-10284
IL-1β antibody	affinity	BF8021
IL-1βELISA kits	Xiamen Lenchangshuo Biotechnology Co., Ltd. (Fujian, China).	ED-10351
IL-6 ELISA kits	Xiamen Lenchangshuo Biotechnology Co., Ltd. (Fujian, China).	ED-10377
ImageJ 1.54 software	NIH
Ipilimumab	MCE	CAS: 477202-00-9
MyD88 antibody	affinity	AF5195
NF-κB p65 antibody	affinity	BF8005
nivolumab	MCE	CAS: 946414-94-4
NLRP3 antibody	affinity	BF8029
Olive oil-based lipid emulsion	the Nutrition Department of Guangzhou Fuda Cancer Hospital
Rapid semi-dry rotary film apparatus	Bio-Rad	788BR04132
Real-time fluorescence qPCR quantitative system	YARUI	MA-6000
RIPA lysis buffer	Beyotime	P0013B
rotary microtome	Leica	RM2235
SDS-PAGE gels	Beyotime	P0052A,P0053A
The Annexin V-FITC/PI Apoptosis Detection Kit	Dalian Meilun Biotechnology Co., Ltd.	MA0220-1
the EasySep Mouse CD4+ T Cell Isolation Kit	Stemcell	19765
The HL-1 rat cardiomyocyte cell line	Guangzhou All-perfect Biotechnology Co., Ltd.	TCM-C783
TNF-α ELISA kits	Xiamen Lenchangshuo Biotechnology Co., Ltd. (Fujian, China).	ED-11776
ultrasound photoacoustic imaging system	Fujifilm VisualSonics	VEvo3100
Urodynamic testing system	Techman	BL-420