Contributions
Abstract: P469
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - 15 Immunology
Background: Multiple sclerosis (MS) is characterized by increased immune activation of peripheral mononuclear cells, but little is known about the specific pathways that are dysregulated. Abnormal levels of phosphorylated proteins and correlations with disease activity and treatment response have been reported in several autoimmune diseases. Phosphoflow is a flow cytometry-based technology that allows tracking multiple intracellular signaling molecules at the single-cell level in response to different stimuli. In this study we aimed to profile the levels of different phosphorylated proteins and phosphorylation response in treatment-naive MS patients.
Methods: We developed a phosphoflow protocol to quantify the levels of 11 phosphorylated proteins
(Btk, Akt, PLCγ, Cbl, p38MAPK, ERK1/2, Stat1, Stat3, Stat4, Stat5 and Stat6), at baseline conditions and after cell activation, in distinct peripheral blood cell populations isolated from 41 treatment-naive relapsing remitting (RRMS) patients and 37 matched controls. Levels of HLA-ABC, HLA-E and HLA-DR were also assessed after stimulation with IFN-α and IFN-γ. A second independent sample set consisted of 9 RRMS and 10 secondary progressive (SP) MS patients.
Results: No significant differences were observed at baseline conditions between patients and controls. However, levels of phosphorylated STAT (p-STAT) proteins were highly upregulated across all cell types in MS patients compared to controls after stimulation with IFN-α. This difference was particularly significant for p-STAT1 and p-STAT6 in the NK cell population (p=2.5x10-6 and p=3.2x10-6 respectively), and for p-STAT1 in monocytes (p=1.2x10-4). Furthermore, levels of all p-STAT proteins correlated with the expression of HLA molecules in monocytes after stimulation with IFN-α, but especially levels of p-STAT1 correlated with expression of HLA-DR (cor=0.6; p=2.8x10−3). On the other hand, the levels of phosphorylated proteins did not differ between RRMS and SPMS patients either in baseline conditions or after stimulation.
Conclusions: We have shown that the response to IFN-α through STAT proteins signaling is strongly dysregulated in MS patients irrespective of disease stage. Furthermore, our findings suggest that the aberrant activation of this pathway could lead to changes in the expression of HLA molecules in NK and antigen presenting cells, which are known to play important roles in the immune response and regulation.
Disclosure:
Ester Canto: nothing to disclose
Stacy J. Caillier: nothing to disclose
Refujia Gomez: nothing to disclose
Stephen L. Hauser: nothing to disclose
Jorge R. Oksenberg: nothing to disclose
Abstract: P469
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - 15 Immunology
Background: Multiple sclerosis (MS) is characterized by increased immune activation of peripheral mononuclear cells, but little is known about the specific pathways that are dysregulated. Abnormal levels of phosphorylated proteins and correlations with disease activity and treatment response have been reported in several autoimmune diseases. Phosphoflow is a flow cytometry-based technology that allows tracking multiple intracellular signaling molecules at the single-cell level in response to different stimuli. In this study we aimed to profile the levels of different phosphorylated proteins and phosphorylation response in treatment-naive MS patients.
Methods: We developed a phosphoflow protocol to quantify the levels of 11 phosphorylated proteins
(Btk, Akt, PLCγ, Cbl, p38MAPK, ERK1/2, Stat1, Stat3, Stat4, Stat5 and Stat6), at baseline conditions and after cell activation, in distinct peripheral blood cell populations isolated from 41 treatment-naive relapsing remitting (RRMS) patients and 37 matched controls. Levels of HLA-ABC, HLA-E and HLA-DR were also assessed after stimulation with IFN-α and IFN-γ. A second independent sample set consisted of 9 RRMS and 10 secondary progressive (SP) MS patients.
Results: No significant differences were observed at baseline conditions between patients and controls. However, levels of phosphorylated STAT (p-STAT) proteins were highly upregulated across all cell types in MS patients compared to controls after stimulation with IFN-α. This difference was particularly significant for p-STAT1 and p-STAT6 in the NK cell population (p=2.5x10-6 and p=3.2x10-6 respectively), and for p-STAT1 in monocytes (p=1.2x10-4). Furthermore, levels of all p-STAT proteins correlated with the expression of HLA molecules in monocytes after stimulation with IFN-α, but especially levels of p-STAT1 correlated with expression of HLA-DR (cor=0.6; p=2.8x10−3). On the other hand, the levels of phosphorylated proteins did not differ between RRMS and SPMS patients either in baseline conditions or after stimulation.
Conclusions: We have shown that the response to IFN-α through STAT proteins signaling is strongly dysregulated in MS patients irrespective of disease stage. Furthermore, our findings suggest that the aberrant activation of this pathway could lead to changes in the expression of HLA molecules in NK and antigen presenting cells, which are known to play important roles in the immune response and regulation.
Disclosure:
Ester Canto: nothing to disclose
Stacy J. Caillier: nothing to disclose
Refujia Gomez: nothing to disclose
Stephen L. Hauser: nothing to disclose
Jorge R. Oksenberg: nothing to disclose