Functional immunogenetics of multiple sclerosis

__Abstract__ In addition to the inflammatory component of MS, neurodegeneration is another important hallmark of the disease. Compared to the immunological component of MS, relatively little is known about the underlying mechanisms of neurodegeneration. Only a few MS risk genes have a presumed function within the CNS and thereby these genes are promising candidates for further study to increase our understanding of neurodegeneration. In chapter 6, we investigated the kinesin kif21b. Kinesins are important proteins involved in the transport of organelles and proteins within cells. These molecules are very important for the survival and function of neurons, since large distances need to be covered. We found that cortical kif21b expression was increased in Alzheimer’s patients (AD, a classical neurodegenerative disease) younger than 62 years compared with MS patients and non-demented controls (NDC). In the white matter, kif21b expression was significantly increased in MS patients compared with NDC. Increasing levels of cortical kif21b were observed in areas with more severe neuropathology in both MS and AD and enhanced kif21b expression was associated with a shorter disease duration. Additionally, accelerated progression to sustained disability (EDSS 6) in MS was found in patients with abundant kif21b expression. Lastly, kif21b protein was expressed in neurons as expected. Interestingly, kif21b protein was also found in astrocytes. Upon astrocyte activation, kif21b expression increased. This may explain the observed differences between the patients groups, as reactive astrocytosis is mainly found in young Alzheimer’s patients and in the white matter of MS patients. Currently, for the minority of MS-associated SNP functional immunogenetic studies have been performed. The majority of studies found functional alterations associated with the SNP. For the remaining SNP, it will be important to perform functional studies to increase our knowledge regarding the (immune)pathogenic mechanism underlying MS, to integrate these studies into clinical trials in order to indentify reliable biomarkers. This will facilitate the development of new more targeted drugs to efficiently treat MS patients and minimising the side effects caused by currently more general immunomodulatory drugs

Elevated EBNA-1 IgG in MS is associated with genetic MS risk variants

Objective: To assess whether MS genetic risk polymorphisms (single nucleotide polymorphism [SNP]) contribute to the enhanced humoral immune response against Epstein-Barr virus (EBV) infection in patients with MS. Methods: Serum anti-EBV nuclear antigen 1 (EBNA-1) and early antigen D (EA-D) immunoglobulin γ (IgG) levels were quantitatively determined in 668 genotyped patients with MS and 147 healthy controls. Anti-varicella-zoster virus (VZV) IgG levels were used as a highly prevalent, non-MS-Associated control herpesvirus. Associations between virus-specific IgG levels and MS risk SNPs were analyzed. Results: IgG levels of EBNA-1, but not EA-D and VZV, were increased in patients with MS compared with healthy controls. Increased EBNA-1 IgG levels were significantly associated with risk alleles of SNP rs2744148 (SOX8), rs11154801 (MYB), rs1843938 (CARD11), and rs7200786 (CLEC16A/CIITA) in an interaction model and a trend toward significance for rs3135388 (HLA-DRB1-1501). In addition, risk alleles of rs694739 (PRDX5/BAD) and rs11581062 (VCAM1) were independently associated and interacted with normal EBNA-1 IgG levels. None of these interactions were associated with EA-D and VZV IgG titers. Conclusions: Several MS-Associated SNPs significantly correlated with differential IgG levels directed to a latent, but not a lytic EBV protein. The data suggest that the aforementioned immune-related genes orchestrate the aberrant EBNA-1 IgG levels

Endotoxin- and ATP-neutralizing activity of alkaline phosphatase as a strategy to limit neuroinflammation

Background: Alkaline phosphatase (AP) is a ubiquitously expressed enzyme which can neutralize endotoxin as well as adenosine triphosphate (ATP), an endogenous danger signal released during brain injury. In this study we assessed a potential therapeutic role for AP in inhibiting neuroinflammation using three complementary approaches.Methods: Mice were immunized to induce experimental autoimmune encephalomyelitis (EAE) and treated with AP for seven days during different phases of disease. In addition, serological assays to determine AP activity, endotoxin levels and endotoxin-reactive antibodies were performed in a cohort of multiple sclerosis (MS) patients and controls. Finally, the expression of AP and related enzymes CD39 and CD73 was investigated in brain tissue from MS patients and control subjects.Results: AP administration during the priming phase, but not during later stages, of EAE significantly reduced neurological signs. This was accompanied by reduced proliferation of splenocytes to the immunogen, myelin oligodendrocyte glycoprotein peptide. In MS patients, AP activity and isoenzyme distribution were similar to controls. Although endotoxin-reactive IgM was reduced in primary-progressive MS patients, plasma endotoxin levels were not different between groups. Finally, unlike AP and CD73, CD39 was highly upregulated on microglia in white matter lesions of patients with MS.Conclusions: Ou

EBNA-1 titer gradient in families with multiple sclerosis indicates a genetic contribution

OBJECTIVE: In multiplex MS families, we determined the humoral immune response to Epstein-Barr virus nuclear antigen 1 (EBNA-1)-specific immunoglobulin γ (IgG) titers in patients with MS, their healthy siblings, and biologically unrelated healthy spouses and investigated the role of specific genetic loci on the antiviral IgG titers. METHODS: IgG levels against EBNA-1 and varicella zoster virus (VZV) as control were measured. HLA-DRB1*1501 and HLA-A*02 tagging single-nucleotide polymorphisms (SNPs) were genotyped. We assessed the associations between these SNPs and antiviral IgG titers. RESULTS: OR for abundant EBNA-1 IgG was the highest in patients with MS and intermediate in their siblings compared with spouses. We confirmed that HLA-DRB1*1501 is associated with abundant EBNA-1 IgG. After stratification for HLA-DRB1*1501, the EBNA-1 IgG gradient was still significant in patients with MS and young siblings compared with spouses. HLA-A*02 was not explanatory for EBNA-1 IgG titer gradient. No associations for VZV IgG were found. CONCLUSIONS: In families with MS, the EBNA-1 IgG gradient being the highest in patients with MS, intermediate in their siblings, and lowest in biologically unrelated spouses indicates a genetic contribution to EBNA-1 IgG levels that is only partially explained by HLA-DRB1*1501 carriership

Cerebrospinal-fluid-derived immunoglobulin G of different multiple sclerosis patients shares mutated sequences in complementarity determining regions

B lymphocytes play a pivotal role in multiple sclerosis pathology, possibly via both antibody-dependent and -independent pathways. Intrathecal immunoglobulin G in multiple sclerosis is produced by clonally expanded B-cell populations. Recent studies indicate that the complementarity determining regions of immunoglobulins specific for certain antigens are frequently shared between different individuals. In this study, our main objective was to identify specific proteomic profiles of mutated complementarity determining regions of immunoglobulin G present in multiple sclerosis patients but absent in healthy controls. To achieve this objective, we purified immunoglobulin G from the cerebrospinal fluid of 29 multiple sclerosis patients and 30 healthy controls and separated the corresponding heavy and light chains via SDS-PAGE. Subsequently, bands were excised, trypsinized, and measured with high-resolution mass spectrometry. We sequenced 841 heavy and 771 light chain variable region peptides. We observed 24 heavy and 26 light chain complementarity determining regions that were solely present in a number of multiple sclerosis patients. Using stringent criteria for the identification of common peptides, we found five complementarity determining regions shared in three or more patients and not in controls. Interestingly, one complementarity determining region with a single mutation was found in six patients. Additionally, one other patient carrying a similar complementarity determining region with another mutation was observed. In addition, we found a skew in the -to-λ ratio and in the usage of certain variable heavy regions that was previously observed at the transcriptome level. At the protein level, cerebrospinal fluid immunoglobulin G shares common characteristics in the antigen binding region among different multiple sclerosis patients. The indication of a shared fingerprint may indicate common antigens for B-cell activation

T cell composition and polygenic multiple sclerosis risk

Background and purpose: Patients with multiple sclerosis (MS) have altered T cell function and composition. Common genetic risk variants for MS affect proteins that function in the immune system. It is currently unclear to what extent T cell composition is affected by genetic risk factors for MS, and how this may precede a possible disease onset. Here, we aim to assess whether an MS polygenic risk score (PRS) is associated with an altered T cell composition in a large cohort of children from the general population. Methods: We included genotyped participants from the population-based Generation R study in whom immunophenotyping of blood T cells was performed at the age of 6 years. Analyses of variance were used to determine the impact of MS-PRSs on total T cell numbers (n = 1261), CD4+ and CD8+ lineages, and subsets therein (n= 675). In addition, T-cell-specific PRSs were constructed based on functional pathway data. Results: The MS-PRS negatively correlated with CD8+ T cell frequencies (p = 2.92 × 10−3), which resulted in a positive association with CD4+/CD8+ T cell ratios (p = 8.27 × 10−9). These associations were mainly driven by two of 195 genome-wide significant MS risk variants: the main genetic risk variant for MS, HLA-DRB1*15:01 and an HLA-B risk variant. We observed no significant associations for the T-cell-specific PRSs. Conclusions: Our results suggest that MS-associated genetic variants affect T cell composition during childhood in the general population.</p

Multiple sclerosis-associated CLEC16A controls HLA class II expression via late endosome biogenesis

C-type lectins are key players in immune regulation by driving distinct functions of antigen-presenting cells. The C-type lectin CLEC16A gene is located at 16p13, a susceptibility locus for several autoimmune diseases, including multiple sclerosis. However, the function of this gene and its potential contribution to these diseases in humans are poorly understood. In this study, we found a strong upregulation of CLEC16A expression in the white matter of multiple sclerosis patients (n = 14) compared to non-demented controls (n = 11), mainly in perivascular leukocyte infiltrates. Moreover, CLEC16A levels were significantly enhanced in peripheral blood mononuclear cells of multiple sclerosis patients (n = 69) versus healthy controls (n = 46). In peripheral blood mononuclear cells, CLEC16A was most abundant in mono