Changes in the plasma proteome profile in LCH patients: identification of differentially expressed proteins
We explored the difference in plasma proteome profiles between 6 patients with MS RO+ LCH and 5 with SS LCH using DIA-based proteomic analysis. Nontargeted proteomics identified 85 DEPs in LCH plasma samples, 27 of which were upregulated and 58 of which were downregulated (fold change >1.5 and adjusted P < 0.05). Volcano plots (Figure 1A) and hierarchical clustering heatmaps (Figure 1B) illustrating the qualified and dysregulated proteins. The expression levels of the top nine up- and downregulated DEPs between the two classifications are shown in Supplementary Figure S1. GO analysis of the biological processes revealed enrichment of differentially expressed proteins involved in the acute-phase response and regulation of immune system processes (Figure 1C). KEGG analysis revealed that complement and coagulation cascades; pathways related to metabolism; and the PI3K-Akt, PPAR, MAPK, and NF-kappa B signaling pathways were significantly enriched (Figure 1D).
Identification of overlapping DEPs/DEGs by scRNA-seq versus plasma proteomics
We next performed a bioinformatics analysis of our previous scRNA-seq dataset from pediatric LCH PBMCs11. We integrated 576 single cells from SS patients and 856 cells from MS RO+ patients to investigate the difference between the two disease extents. Nine cell clusters were identified by tSNE visualization (Figure 2A). We identified 322 differentially expressed genes (DEGs).
Furthermore, we identified three overlapping DEPs/DEGs, CSF1R, CD14, and GSN, between the scRNA-seq and plasma proteomics data (Figure 2B). The differential expression of the three DEGs in the single-cell transcriptome of LCH PBMCs is shown in Figure 2C. Among the three candidates, CD14 is often considered a marker of classical monocyte cells, and GSN was found to be downregulated in the plasma of MS RO+ LCH patients. We focused on CSF1R for further analysis because it plays a critical role in the pathogenesis of LCH.