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 limite....

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 ....

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