Cerebrospinal Fluid Biomarkers in Differential Diagnosis of Multiple Sclerosis and Systemic Inflammatory Diseases with Central Nervous System Involvement.

BACKGROUND: Diagnosis of multiple sclerosis (MS) is established on criteria according to clinical and radiological manifestation. Cerebrospinal fluid (CSF) analysis is an important part of differential diagnosis of MS and other inflammatory processes in the central nervous system (CNS). METHODS: In total, 242 CSF samples were collected from patients undergoing differential MS diagnosis because of the presence of T2-hyperintensive lesions on brain MRI. The non-MS patients were subdivided into systemic inflammatory diseases with CNS involvement (SID) or cerebrovascular diseases (CVD) or other non-inflammatory diseases (NID). All samples were analyzed for the presence of oligoclonal bands and ELISA was performed for detection of: INF gamma, IL-6, neurofilaments light chain (NF-L), GFAP, CHI3L1, CXCL13, and osteopontin. RESULTS: The level of IL-6 (p = 0.024), osteopontin (p = 0.0002), and NF-L (p = 0.002) was significantly different among groups. IL-6 (p = 0.0350) and NF-L (p = 0.0015) level was significantly higher in SID compared to NID patients. A significantly higher level of osteopontin (p = 0.00026) and NF-L (p = 0.002) in MS compared to NID population was noted. ROC analysis found weak diagnostic power for osteopontin and NFL-L. CONCLUSIONS: The classical and non-standard markers of inflammatory process and neurodegeneration do not allow for sufficient differentiation between MS and non-MS inflammatory CNS disorders. Weak diagnostic power observed for the osteopontin and NF-L needs to be further investigated.

NK Cell Induced T Cell Anergy Depends on GRAIL Expression.

NK cells (natural killer cells) being a part of the innate immune system have been shown to be involved in immunoregulation of autoimmune diseases. Previously we have shown that HINT1/Hsp70 treatment induced regulatory NK cells ameliorating experimental autoimmune encephalomyelitis (EAE) course and CD4+ T cells proliferation. NK cells were isolated from mice treated with HINT1/Hsp70 and co-cultured with proteolipid protein (PLP)-stimulated CD4+ T cells isolated from EAE mice. Cell proliferation was assessed by thymidine uptake, cytotoxicity by lactate dehydrogenase (LDH) release assay and fluorescence activated cell sorting (FACS) analysis, protein expression by Western blot, mRNA by quantitative RT-PCR. Gene related to anergy in lymphocytes (GRAIL) expression was downregulated by specific siRNA and GRAIL overexpression was induced by pcDNA-GRAIL transfection. HINT1/Hsp70 pretreatment of EAE SJL/J mice ameliorated EAE course, suppressed PLP-induced T cell proliferation by enhancing T cell expression of GRAIL as GRAIL downregulation restored T cell proliferation. HINT1/Hsp70 treatment induced immunoregulatory NK cells which inhibited PLP-stimulated T cell proliferation not depending on T cell necrosis and apoptosis. This immunoregulatory NK cell function depended on NK cell expression of GRAIL as GRAIL downregulation diminished inhibition of NK cell suppression of T cell proliferation. Similarly GRAIL overexpression in NK cells induced their regulatory function. HINT1/Hsp70 treatment generated regulatory NK cells characterized by expression of GRAIL.

Multiple sclerosis: Serum-derived exosomes express myelin proteins.

BACKGROUND: Exosomes are small extracellular vesicles that provide cell-to-cell communication and are involved in immunoregulation. OBJECTIVE: To investigate serum exosomes for the presence of myelin proteins outside the central nervous system (CNS) and their role in multiple sclerosis (MS). METHODS: Serum, cerebrospinal fluid (CSF), and peripheral blood mononuclear cell (PBMC) samples were collected from 45 patients with relapsing-remitting MS (RRMS), 30 patients with secondary progressive MS (SPMS), and 45 healthy controls. Exosomes were isolated using a polymer formulation method, and their size, concentration, and CNS myelin protein contents were measured by a nanoparticle tracking analysis, enzyme-linked immunosorbent assays, and Western blot. RESULTS: We found that exosomes expressed three major myelin proteins, myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein (MOG). Exosomal content of MOG strongly correlated with disease activity and was highest in RRMS patients in relapse and in SPMS patients. Serum-derived exosomes induced proliferation of MOG-T cell receptor transgenic T cells confirming that serum exosomes maintained MOG immunogenicity. CONCLUSION: Exosomes isolated outside CNS tissue expressed myelin proteins, and the presence of MOG correlated strongly with disease activity. We conclude that exosomes might enhance and/or perpetuate anti-myelin immune reactions in MS and may provide novel markers of disease activity.

Multiple sclerosis: Presence of serum antibodies to lipids and predominance of cholesterol recognition.

A lot of available data on lipid immunology in multiple sclerosis (MS) have been derived from studies using synthetic lipids, therefore the role of lipids in the immunopathogenesis of MS remains poorly defined. The present study on the lipid response in MS was performed on native lipids from autopsied brain tissue. For this, lipid fractions (n = 9) were prepared from MS (n = 3) and control (n = 2) white matter according to the Folch procedure and were characterized depending on their solubility in chloroform/methanol. TLC showed that, in brain from MS cases, neutral lipids were rich in cholesterol and cholesterol esters while lipids from control brains displayed a predominance of phospholipids. MS serum IgG and IgM were found to bind to MS brain lipid fractions with a higher efficacy (p < 0.05) than the control serum. F(ab)2 fractionation revealed that MS serum IgG binding depended on a specific antibody-type of recognition. Pre-adsorption of serum with cholesterol, galactocerebrosides, sulfitides, and phosphatidylinositol prior to ELISA with MS brain lipids, showed that cholesterol diminished IgG and IgM binding up to 70%. Experiments with synthetic lipids confirmed the predominance of cholesterol binding by MS serum. Our results demonstrate that IgG and IgM fractions from MS serum specifically and predominantly recognize native cholesterol and cholesterol esters isolated from the brain tissue of patients with MS. © 2017 Wiley Periodicals, Inc.

Global exosome transcriptome profiling reveals biomarkers for multiple sclerosis.

OBJECTIVE: Accumulating evidence supports a role for exosomes in immune regulation. In this study, we investigated the total circulating exosome transcriptome in relapsing-remitting multiple sclerosis (RRMS) patients and healthy controls (HC). METHODS: Next generation sequencing (NGS) was used to define the global RNA profile of serum exosomes in 19 RRMS patients (9 in relapse, 10 in remission) and 10 HC. We analyzed 5 million reads and >50,000 transcripts per sample, including a detailed analysis of microRNAs (miRNAs) differentially expressed in RRMS. The discovery set data were validated by quantification using digital quantitative polymerase chain reaction with an independent cohort of 63 RRMS patients (33 in relapse, 30 in remission) and 32 HC. RESULTS: Exosomal RNA NGS revealed that of 15 different classes of transcripts detected, 4 circulating exosomal sequences within the miRNA category were differentially expressed in RRMS patients versus HC: hsa-miR-122-5p, hsa-miR-196b-5p, hsa-miR-301a-3p, and hsa-miR-532-5p. Serum exosomal expression of these miRNAs was significantly decreased during relapse in RRMS. These miRNAs were also decreased in patients with a gadolinium enhancement on brain magnetic resonance imaging. In vitro secretion of these miRNAs by peripheral blood mononuclear cells was also significantly impaired in RRMS. INTERPRETATION: These data show that circulating exosomes have a distinct RNA profile in RRMS. Because putative targets for these miRNAs include the signal transducer and activator of transcription 3 and the cell cycle regulator aryl hydrocarbon receptor, the data suggest a disturbed cell-to-cell communication in this disease. Thus, exosomal miRNAs might represent a useful biomarker to distinguish multiple sclerosis relapse. Ann Neurol 2017;81:703-717.

HINT1 peptide/Hsp70 complex induces NK-cell-dependent immunoregulation in a model of autoimmune demyelination.

Heat shock proteins (Hsps) interact with the immune system and have been shown to contribute to immunoregulation. As efficient chaperones, Hsps bind many peptides and these complexes have many yet-to-be-clarified functions. We have shown that Hsp70 is complexed within the mouse CNS with peptide CLAFHDISPQAPTHFLVIPK derived from histidine triad nucleotide-binding protein-1 (HINT138₋57/Hsp70). Only this complex, in contrast to other peptides complexed with Hsp70, was able to prevent experimental autoimmune encephalomyelitis (EAE) by induction of immunoregulatory mechanisms dependent on NK cells. Pretreatment of proteolipid protein peptide 139₋151(PLP139₋151) sensitized SJL/J mice with HINT138₋57/Hsp70 prevented the development of EAE, suppressed PLP139₋151-induced T-cell proliferation, and blocked secretion of IL-17. HINT138₋57 /Hsp70 stimulation of NK cells depended on synergistic activation of two NK-cell receptors, CD94 and NKG2D. NK cells with depleted CD94 or with blocked NKG2D did not inhibit PLP139₋151-induced spleen cell (SC) proliferation. The HINT138₋57/Hsp70 complex enhanced surface expression of the NKG2D ligand-H60. Downstream signaling of CD94 and NKG2D converged at the adaptor proteins DAP10 and DAP12, and in response to HINT138₋57 /Hsp70 stimulation, expression of DAP10 and DAP12 was significantly increased in NK cells. Thus, we have shown that the HINT138₋57 /Hsp70 complex affects NK-cell function by enhancing NK-cell-dependent immunoregulation in the EAE model of autoimmune demyelination.

EAE tolerance induction with Hsp70-peptide complexes depends on H60 and NKG2D activity.

Inflammation leads to induction of tissue stress conditions that might contribute to the generation of mechanisms limiting ongoing immune responses. We have shown previously that peptides derived from brain tissue of mice with experimental autoimmune encephalomyelitis (EAE) complexed with the chaperone heat shock protein 70 (Hsp70-pc) induce an NK-cell-dependent tolerance for subsequent EAE sensitization. We now present data that showed that the MHC class I-related glycoprotein H60 determines Hsp70-pc-induced EAE inhibition. Hsp70-pc led to significant and selective up-regulation of H60 expression in SJL/J mice, and Ab-blocking of H60 expression led to loss of EAE tolerance. Similarly, blocking of the NK cell receptor for H60, NKG2D, also reversed the Hsp70-pc-induced EAE inhibition. In contrast, in C57BL/6 mice H60 was not expressed, and Hsp70-pc-induced tolerance was not detected. The NK cell mediated Hsp70-pc-induced tolerance to EAE was dependent on modulation of dendritic cells function leading to diminished T cell reactivity to PLP. As, no increase of H60 expression on T cells from EAE mice immunized with PLP was detected, and no enhanced loss of CD3+ H60+ over CD3+ H60- cells in Hsp70-pc-induced EAE tolerance was found direct killing of H60+ PLP-reactive cells seems not to be involved in the Hsp70-pc-induced tolerance induction. We have provided evidence that Hsp70-pc-induced tolerance for EAE, mediated by NK cells, involves induction of H60 ligand and its interaction with NKG2D receptor. NK cells tolerization of EAE depends on altered dendritic cells activity leading to enhanced death of Ag reactive cells.

Brain-derived heat shock protein 70-peptide complexes induce NK cell-dependent tolerance to experimental autoimmune encephalomyelitis.

Heat shock proteins (Hsp) are markedly up-regulated at sites of inflammation during autoimmune diseases like experimental autoimmune encephalomyelitis (EAE). In this study, we show that Hsp70-peptide complexes (pc) isolated from brains of mice with EAE prevented the development of EAE clinically and pathologically when administered before proteolipid protein 139-151 (PLP139-151) immunization. In contrast, pure Hsp70 or Hsp70-pc derived from brains of healthy mice or other inflamed tissue did not modulate the expression of EAE. In animals in which EAE had been suppressed by Hsp70-pc, lymphocytes showed increased cell death in response to PLP139-151 that correlated with elevated IFN-gamma and NO production. Coculture of spleen cells from Hsp70-pc immunized mice with spleen cells from untreated EAE mice, in addition to depletion experiments, showed that NK cells reduced reactivity to PLP139-151. Transfer of NK cells from Hsp70-pc-immunized mice to recipients sensitized for EAE abolished disease development. Thus, we propose that Hsp70 demonstrate the ability to bind to peptides generated during brain inflammation and to induce a regulatory NK cell population that is capable of preventing subsequent autoimmunization for EAE.