MAPK pathway and B cells overactivation in multiple sclerosis revealed by phosphoproteomics and genomic analysis

Abstract

Dysregulation of signaling pathways in multiple sclerosis (MS) can be analyzed by phosphoproteomics in peripheral blood mononuclear cells (PBMCs). We performed in vitro kinetic assays on PBMCs in 195 MS patients and 60 matched controls and quantified the phosphorylation of 17 kinases using xMAP assays. Phosphoprotein levels were tested for association with genetic susceptibility by typing 112 single-nucleotide polymorphisms (SNPs) associated with MS susceptibility. We found increased phosphorylation of MP2K1 in MS patients relative to the controls. Moreover, we identified one SNP located in the PHDGH gene and another on IRF8 gene that were associated with MP2K1 phosphorylation levels, providing a first clue on how this MS risk gene may act. The analyses in patients treated with disease-modifying drugs identified the phosphorylation of each receptor's downstream kinases. Finally, using flow cytometry, we detected in MS patients increased STAT1, STAT3, TF65, and HSPB1 phosphorylation in CD19⁺ cells. These findings indicate the activation of cell survival and proliferation (MAPK), and proinflammatory (STAT) pathways in the immune cells of MS patients, primarily in B cells. The changes in the activation of these kinases suggest that these pathways may represent therapeutic targets for modulation by kinase inhibitors.

Keywords: B cells; autoimmunity; multiple sclerosis; phosphoproteomics; signaling pathways.

Fig. 1.

The immune signaling network in MS. Shown are the stimuli used for the in vitro assays (purple hexagons on the left), targeting receptors on the cell membrane (orange circles), although some stimuli directly target kinases or pathways (e.g., H₂O₂ directly induces oxidative stress). Membrane receptors are linked to intracellular kinases (light grey circles) as part of their intracellular signaling networks. The specific phosphoproteins tested in these assays are shown as red circles associated to the master kinase. Finally, kinases influence cellular and molecular processes. Stimulation (e.g., phosphorylation) is indicated by green arrows, whereas inhibitory interactions (e.g., dephosphorylation) is indicated by a red T link.

Fig. 2.

MP2K1 phosphorylation in PBMCs from MS patients and controls. Phosphorylation of MP2K1 in PBMCs from MS patients and controls, as assessed by xMAP and compared with a Wilcoxon test: *P < 0.05; **adjusted P < 0.05. n.s., not significant.

Fig. 3.

The phosphoproteomic signature in MS. The lines in the circle graph show the pairs of stimuli/phosphoproteins found to be significant and the corresponding MS susceptibility genes (ANOVA test for association between kinase levels and SNPs; Benjamini correction for multiple testing). The outer circle shows the 17 phosphoproteins analyzed and the 19 stimuli used, while the inner circle shows the 112 SNPs tested (color coded for the mean allelic distribution) where the SNP indicated by the link is referred to by its letter code. (A) Phosphoproteomic signature in MS, healthy controls, and the RMS and PMS subtypes of MS. (B) Phosphoproteomic signature of the DMDs compared with untreated RRMS patients.

Fig. 4.

Protein network analysis of phosphoproteins and SNPs associated with MS. The graph shows the proteins from either genes containing MS-susceptibility SNPs (in red) or phosphokinases (in blue) found associated in our analysis. (A) First-order physical interactions of the studied proteins identified on the iRefIndex and MetaBase/MetaCore database. (B) Transcriptional regulation (dashed lines) and physical interactions (solid lines) of the associated proteins identified using the TieDie algorithm on the background of directed MetaBase network.