Multi-Omics Integration Analysis Identifies Lipoproteins As Biomarkers for Glucocorticoid Response in Immune Thrombocytopenia

Introduction

Glucocorticoids (GCs) are the first-line therapy for most immune diseases due to their high effectiveness in immune suppression, including immune thrombocytopenia (ITP). However, some newly diagnosed ITP patients exhibit low responsiveness to GCs, necessitating second-line therapies which increase both the financial burden and bleeding risk. The mechanisms behind glucocorticoid resistance in ITP remain incompletely understood. Here, we explore differences in the CD4⁺ T-cell proteomic and transcriptomic profiles, as well as the plasma proteomic profiles, between newly diagnosed ITP patients and healthy individuals, as well as GCs-sensitive and GCs-resistant ITP patients.

Methods

We prospectively recruited 13 newly diagnosed ITP patients and 5 volunteers as healthy controls (HC). Patients received guideline recommended glucocorticoid monotherapy (methylprednisolone 0.8-1 mg/kg/day) and peripheral blood samples were collected before administration. Based on the therapeutic effect (platelet count > 30×10^9/L or < 30×10^9/L) two weeks after glucocorticoid administration, patients were divided into a GC-sensitive group (GCS, n=9) and a GC-resistant group (GCR, n=4). Plasma was isolated for comprehensive 4D-DIA mass spectrometry. CD4⁺ T-cells were isolated from fresh peripheral blood samples using immunomagnetic beads, achieving a purity of over 90%, and utilized for Tandem Mass Tag protein quantitation and bulk RNA sequencing.

Results

In newly diagnosed ITP patients, 30 up-regulated and 5 down-regulated proteins in plasma were identified. Compared with HC, the level of apolipoprotein E in plasma was significantly elevated in ITP patients, with even higher level in the GCR group, accompanied by increased cholesteryl ester transfer protein. Lipid droplet staining by BODIPY 493/503 and flow cytometry showed that ITP patients had lower lipid droplet fluorescence intensity, indicating lower triglyceride storage in CD4⁺ T-cells. Serum lipid tests also showed that high-density lipoprotein levels were increased in GCR compared with GCS. This suggested that lipid metabolism may be more significantly altered in GCR patients.

We further investigated the transcriptomic profiles of CD4⁺ T-cells. KEGG and GO enrichment analysis revealed that CD4⁺ T-cells from ITP showed activated MAPK cascade, IL-17 signaling pathway, and atherosclerosis. However, the plasma membrane signaling receptor complex was decreased, suggesting interruption in signal transduction. Then, 28 up-regulated and 21 down-regulated genes were detected in GCR. No significant differences in NR3C1, P-glycoprotein, or lipid metabolism associated gene were observed.

As for cellular proteomic, 1496 different expressed proteins were evaluated in ITP patients, with highly increased apolipoprotein C. GO and KEGG analysis indicated that ITP patients had lower oxidative phosphorylation, higher glycolysis, increased cellular lipid biosynthesis, and higher lipid kinase activity, supporting metabolic reprogramming in ITP. In total, 167 up-regulated and 132 down-regulated proteins were observed in T-cells from GCR, associated with lower apoptosis and activated HIF-1 signaling pathway. Finally, we established a PPI network of the different expression proteins between GCR and GCS patients, identifying forkhead box protein O3 (FOXO3) and proto-oncogene tyrosine-protein kinase (Src) as hub proteins.

Conclusion

Our findings indicate that lipid metabolism could become a potential pathogenesis of ITP and a target for overcoming glucocorticoid resistance in autoimmune diseases. Apolipoprotein E could become a new biomarker for detecting glucocorticoid resistant ITP patients. Additionally, metabolism reprogramming in CD4⁺ T-cells might be associated with Src family kinase and downstream pathways, leading to glucocorticoid resistance.