Proteomic Profiles of Neutrophils from Behcet's Uveitis Patients and their Sex Differences

Abstract

Behcet's uveitis (BU) is one of the most vision-threatening uveitis entities with male-biased incidence and severity. Neutrophil dysfunction has been implicated in the pathogenesis of this disease. However, their proteomic changes are not completely understood. We performed proteomic analysis on peripheral neutrophils from patients with active BU and identified 82 up-regulated and 516 down-regulated differentially expressed proteins (DEPs) compared to healthy controls (HCs). We further performed functional analysis on these DEPs and found that the pathway involved in neutrophil extracellular trap formation was activated, whereas nucleotide metabolism and apoptosis were suppressed. Compared with female patients, male patients presented enhanced pathways associated neutrophil-mediated inflammatory responses and suppressed apoptosis. Additionally, integrative analysis of proteomic profiles and single-cell RNA sequencing (scRNA-seq) data revealed that these sex differences might be related to the enhanced inflammatory response in primed inflammatory and inflammatory neutrophils as well as deficiencies in apoptosis and nucleotide metabolism in ROS-responsive neutrophils. Collectively, our data revealed the proteomic profiles of neutrophils from patients with BU, and their functional changes may play crucial roles in the pathogenesis of this disease and its sex differences.

Keywords: Apoptosis; Behcet's uveitis; Neutrophil hyperactivation; Nucleotide metabolism; Proteomics.

Fig. 1

Proteomic profiling of neutrophils from patients with active BU. A The workflow of integrative analysis on neutrophils from BU patients and HCs. B Principal component  analysis (PCA) between BU patients (N = 9) and HCs (N = 9). C Volcano plot representing the DEPs between BU patients and HCs. Proteins exhibiting a fold change > 1.5 and p.adjust < 0.05 (Benjamini-Hochberg method) were classified as DEPs. Red dots indicate significantly up-regulated proteins, blue dots indicate significantly down-regulated proteins, and gray dots represent proteins with no significant difference

Fig. 2

Functional analysis of the DEPs between BU patients and HCs. A GO enrichment analysis (BP) of up-regulated and down-regulated DEPs between BU patients and HCs. B PPI analysis of DEPs between BU patients and HCs. Pagerank algorithm was used to select the top 50 vertices to build the network. The PPI networks were constructed using the STRING database. C KEGG enrichment analysis of the DEPs between BU patients and HCs. D KEGG enrichment analysis of up-regulated and down-regulated DEPs between BU patients and HCs. E GSEA analysis on the proteomic profiling of neutrophils between BU patients and HCs

Fig. 3

Sex differences in neutrophil proteomes of BU patients. A Volcano plot representing the DEPs between male BU patients and female BU patients. Proteins exhibiting a fold change > 1.5 and p.adjust < 0.05 (Benjamini-Hochberg method) were classified as DEPs. Red dots indicate significantly up-regulated proteins, blue dots indicate significantly down-regulated proteins, and gray dots represent proteins with no significant difference. B PPI analysis of DEPs between male BU patients and female BU patients. Pagerank algorithm was used to select the top 50 vertices to build the network. The PPI networks were constructed using the STRING database. C KEGG enrichment analysis of DEPs between male BU patients and female BU patients. D KEGG enrichment analysis of up-regulated and down-regulated DEPs between male BU patients and female BU patients. E GSEA analysis was used to assess whether the pathway was up-regulated or down-regulated

Fig. 4

Integrative analysis of the sex-specific proteomic profiles and scRNA-seq data of neutrophils from BU patients. A Venn diagrams representing the shared DEGs between proteomic profiles and neutrophil subsets annotated in the scRNA-seq dataset. B Differential expression of shared DEGs in each subset of neutrophils. C GO enrichment analysis (BP) of shared DEPs between male and female BU patients