Cholesterol and Stearamide Mediate Lymphocyte Apoptosis and Cytokine Secretion in Systemic Lupus Erythematosus

Summary

Here, we present a study that integrates two-sample Mendelian randomization, liquid chromatography-mass spectrometry (LC-MS)-based metabolomics validation, and functional assays using Systemic Lupus Erythematosus (SLE) patient-derived lymphocytes to assess apoptosis and cytokine responses following metabolite stimulation, aiming to identify lipid-related biomarkers and investigate their immunoregulatory roles in SLE.

Abstract

Alterations in serum metabolite composition have been increasingly associated with systemic lupus erythematosus (SLE), yet the direct effects of these metabolites on immune cell function remain poorly defined. This study aimed to identify peripheral blood metabolites associated with SLE and evaluate their impact on apoptosis and cytokine secretion in lymphocytes derived from SLE patients.

We performed a two-sample Mendelian randomization (MR) analysis to investigate the relationship between 565 serum metabolites and SLE, using the inverse-variance weighted model as the primary analytical approach. Significant findings were cross-validated using previously published untargeted metabolomics data based on liquid chromatography-mass spectrometry (LC-MS). In functional experiments, lymphocytes isolated from SLE patients were stimulated with target metabolites, followed by assessment of apoptosis and cytokine secretion.

MR analysis identified 28 metabolites significantly associated with SLE. Of these, cholesterol (OR = 1.462, 95% CI: 1.100-1.940, P = 0.008) and stearamide (OR = 0.125, 95% CI: 0.020-0.660, P = 0.014) were validated through LC-MS and found to be elevated in SLE patients. Receiver operating characteristic analysis demonstrated strong diagnostic performance (AUC = 0.999 for cholesterol; AUC = 1.000 for stearamide). Functionally, both metabolites induced increased apoptosis and elevated secretion of TNF-α, IFN-γ, and TGF-β1 in SLE lymphocytes.

In summary, our integrated approach combining genetic association, metabolite profiling, and functional assays reveals that cholesterol may contribute to immune dysregulation in SLE. Although stearamide showed a negative association with SLE risk in MR analysis, its in vitro effects mirrored those of cholesterol, suggesting a complex and context-dependent role. These findings underscore the importance of lipid metabolism in SLE and support further mechanistic and clinical investigation of its immunological impact.

Introduction

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease, distinguished by complex pathogenesis and diverse clinical manifestations, that affects millions of people worldwide¹. The heterogeneity of SLE-where symptoms differ widely among individuals and evolve over time-creates significant challenges in diagnosis, monitoring disease activity, and tailoring personalized treatment plans^2,3. Although the 2019 European League Against Rheumatism and American College of Rheumatology (EULAR/ACR) SLE classification criteria have improved diagnostic accuracy⁴, th....

Protocol

​This study was conducted per the 2019 EULAR/ACR classification criteria. This study was approved by Shenzhen People's Hospital (Ethics Committee Approval Number LL-KY-2019514).

Mendelian randomization (MR) analysis

GWAS data sources
Genetic association data for SLE and 565 metabolites were obtained from the OpenGWAS database (https://gwas.mrcieu.ac.uk/). The disease GWAS datasets are summarized in Table 1, while the sources and sample sizes for the metabolite GWAS data are detailed in Table 2. These datasets provided the fou....

Results

Association between key metabolites and clinical manifestations in SLE

In this study, MR analysis of 565 metabolites with SLE identified significant causal associations. As shown in Figure 2, a total of 28 metabolites significantly associated with SLE were identified, encompassing categories such as peptides, amino acids, energy, lipids, carbohydrates, and fatty acids. Peptides such as gamma-glutamylphenylalanine and pro-hydroxy-pro, amino acids i.......

Discussion

Our study demonstrates that 1.5 mmol/L cholesterol, 5 µmol/L stearamide, and 10 µmol/L stearamide induce similar patterns of apoptosis and cytokine secretion in lymphocytes from SLE patients, although variations in magnitude were observed. Specifically, both 1.5 mmol/L cholesterol and 5 µmol/L stearamide significantly increased apoptosis in SLE lymphocytes. Additionally, treatment with 1.5 mmol/L cholesterol in combination with either 5 µmol/L or 10 µmol/L stearamide further enhanced the secretio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Annexin V-FITC/PI Apoptosis Kit	Multi Sciences	AP101	For apoptosis detection via flow cytometry
Cholesterol	Shanghai Jinpan Biotech Co., Ltd.	JP1210051	Used for stimulation experiments in PBMCs (10 g)
Counterwell Insert	Corning	CBG10001	Used to physically separate cells in coculture experiments
Flow Cytometer	Beckman Coulter	CytoFLEX	Flow cytometric analysis
GraphPad Prism Software	GraphPad		Statistical analysis and graphing
LEGENDplex Human Essential Immune Response Panel	BioLegend	740390	Multiplex detection of immune-related cytokines
Micropipette Set	Eppendorf	Various	Liquid handling and volume transfer (0.1–10 μL, 10–100 μL, 100–1000 μL)
Peripheral Blood Lymphocyte Separation Medium	Domestic Manufacturer	B1.1420A	Used for PBMC isolation (200 mL)
RPMI-1640 Medium	Gibco	C22400500BT	Culture medium for PBMCs
Stearamide	Shanghai Jinpan Biotech Co., Ltd.	1267285	Used for lipid stimulation of PBMCs (100 g)
Sterile 24-well Plate	Domestic Manufacturer	BLJ182A	For cell culture
CountStar	Shanghai Ruiyu Biotechnology	CS200	Automatic cell counter
PBS	Hyclone	10010023	Cell washing
Ficoll-Paque	Cytiva	17-5442-03	Synovial cell isolation
LEGENDplex software	BioLegend	Version 8	Flow cytometry data analysis
GraphPad Prism 9.0	GraphPad Software	Version 9	Statistical analysis