Ekaterina Kotelnikova, Narsis A. Kiani, Dimitris Messinis, Inna Pertsovskaya, Vicky Pliaka, Marti Bernardo-Faura, Melanie Rinas, Gemma Vila, Irati Zubizarreta, Irene Pulido-Valdeolivas, Theodore Sakellaropoulos, Wolfgang Faigle, Gilad Silberberg, Mar Masso, Pernilla Stridh, Janina Behrens, Tomas Olsson, Roland Martin, Friedemann Paul, Leonidas G. Alexopoulos, Julio Saez-Rodriguez, Jesper Tegner, and Pablo Villoslada

PNAS May 7, 2019 116 (19) 9671-9676

Significance

The alteration of cell communication in the immune system of patients with multiple sclerosis (MS) can be analyzed by phosphoproteomics. We performed in vitro assays on immune cells from MS patients and controls and quantified the phosphorylation of several kinases. We found increased phosphorylation of several MAPK kinases in patients, which were modulated by several genetic markers associated with the disease. By using flow cytometry, we detected several kinases altered by phosphorylation in B 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.

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.

See: https://www.pnas.org/content/116/19/9671

Figure 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.