Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by profound metabolic perturbations and dysregulated crosstalk between immune cells and fibroblast-like synoviocytes (FLS). Phenylalanine (PHE), an aromatic amino acid, has been implicated in RA-related metabolic disorders, but its direct immunomodulatory effects on human primary FLS and peripheral blood lymphocytes remain unclear. This study aimed to identify RA-specific metabolic signatures via untargeted metabolomics and validate PHE’s functional role in regulating core pathogenic cells in RA. Methods LC–MS/MS-based untargeted metabolomics was performed on serum from 123 RA patients (2010 ACR/EULAR criteria) and 106 age/sex-matched healthy controls. Differential metabolites and pathways were identified via multivariate statistical analysis (PCA, OPLS-DA) and KEGG enrichment. Primary FLS and peripheral blood lymphocytes were treated with 100 μmol/L PHE (matching RA serum levels). CCK-8, qPCR, multiplex cytokine detection and flow cytometry were used to assess cell viability, pro-inflammatory gene expression, cytokine secretion, and apoptosis. Results Metabolomic profiling revealed clear separation between RA and controls, with significant enrichment in phenylalanine metabolism. Eight novel RA-specific biomarkers were identified. Functional validation showed that PHE significantly upregulated mRNA expression of IL-6, IL-8, CXCL12, and MMP3 in FLS (1.8–7.2-fold, all p < 0.01) and enhanced secretion of IL-6 (115 → 180 pg/mL), IL-8 (300 → 550 pg/mL), and CCL2 (3000 → 5500 pg/mL) (all p < 0.01). In lymphocytes, PHE promoted secretion of IL-1β, TNF-α, IL-6, and CXCL8 (all p < 0.001) and increased total apoptosis rate (8.3% → 18.7%, p < 0.01). Conclusion This study reveals distinct metabolic features in RA patients, confirming PHE as a key pro-inflammatory metabolite that modulates FLS and lymphocyte functions.<Table Float="No" ID="Taba"> <tgroup cols="3"> <colspec align="left" colname="c1" colnum="1" /> <colspec align="left" colname="c2" colnum="2" /> <colspec align="left" colname="c3" colnum="3" /> <tbody> <row> <entry nameend="c3" namest="c1"> Key Points • Serum untargeted metabolomics of an RA cohort identifies 8 novel biomarkers and core dysregulated pathways, including phenylalanine metabolism and acylcarnitine-mediated fatty acid β-oxidation. • Phenylalanine directly activates FLS, upregulating pro-inflammatory cytokines, chemokines, and matrix metalloproteinases, enhancing their tissue-destructive potential. • Phenylalanine promotes pro-inflammatory cytokine secretion and induces apoptosis in RA peripheral blood lymphocytes, disrupting immune homeostasis. </entry> </row> </tbody> </tgroup> </Table>

Metabolomics insights into RA: the proinflammatory role of phenylalanine in human FLS and primary lymphocytes

期刊论文
发表日期：

2026年4月15日

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Jinquan Shen,
Xu Gao,
WeiYe Pan,
ZhengYu Fang,
XiaoCheng Wang,
Qingwen Wang

摘要

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by profound metabolic perturbations and dysregulated crosstalk between immune cells and fibroblast-like synoviocytes (FLS). Phenylalanine (PHE), an aromatic amino acid, has been implicated in RA-related metabolic disorders, but its direct immunomodulatory effects on human primary FLS and peripheral blood lymphocytes remain unclear. This study aimed to identify RA-specific metabolic signatures via untargeted metabolomics and validate PHE’s functional role in regulating core pathogenic cells in RA.

Methods

LC–MS/MS-based untargeted metabolomics was performed on serum from 123 RA patients (2010 ACR/EULAR criteria) and 106 age/sex-matched healthy controls. Differential metabolites and pathways were identified via multivariate statistical analysis (PCA, OPLS-DA) and KEGG enrichment. Primary FLS and peripheral blood lymphocytes were treated with 100 μmol/L PHE (matching RA serum levels). CCK-8, qPCR, multiplex cytokine detection and flow cytometry were used to assess cell viability, pro-inflammatory gene expression, cytokine secretion, and apoptosis.

Results

Metabolomic profiling revealed clear separation between RA and controls, with significant enrichment in phenylalanine metabolism. Eight novel RA-specific biomarkers were identified. Functional validation showed that PHE significantly upregulated mRNA expression of IL-6, IL-8, CXCL12, and MMP3 in FLS (1.8–7.2-fold, all p < 0.01) and enhanced secretion of IL-6 (115 → 180 pg/mL), IL-8 (300 → 550 pg/mL), and CCL2 (3000 → 5500 pg/mL) (all p < 0.01). In lymphocytes, PHE promoted secretion of IL-1β, TNF-α, IL-6, and CXCL8 (all p < 0.001) and increased total apoptosis rate (8.3% → 18.7%, p < 0.01).

Conclusion

This study reveals distinct metabolic features in RA patients, confirming PHE as a key pro-inflammatory metabolite that modulates FLS and lymphocyte functions.

Key Points

• Serum untargeted metabolomics of an RA cohort identifies 8 novel biomarkers and core dysregulated pathways, including phenylalanine metabolism and acylcarnitine-mediated fatty acid β-oxidation.

• Phenylalanine directly activates FLS, upregulating pro-inflammatory cytokines, chemokines, and matrix metalloproteinases, enhancing their tissue-destructive potential.

• Phenylalanine promotes pro-inflammatory cytokine secretion and induces apoptosis in RA peripheral blood lymphocytes, disrupting immune homeostasis.