Single-cell multiomics in neuroinflammation.

The central nervous system (CNS) is, more than other organs, particularly vulnerable to inflammation and immune responses must be tightly controlled in order to maintain host protection. Accordingly, neuroinflammation is an orchestrated process involving various cell types that may dramatically change their phenotypic and functional properties upon entering the CNS. Recent advances in single-cell multiomics offer the unique opportunity to resolve this cellular heterogeneity in a holistic fashion and reshape our understanding of the molecular and cellular processes during neuroinflammation. Here, we provide an overview of technical advances in single-cell multiomics and the tremendous impact on our basic understanding of neuroinflammation. We discuss insights obtained in neuroinflammatory diseases and elaborate to which extent these tool sets could be applied in a clinical setting.

Plasma lipidomics of monozygotic twins discordant for multiple sclerosis.

Blood biomarkers of multiple sclerosis (MS) can provide a better understanding of pathophysiology and enable disease monitoring. Here, we performed quantitative shotgun lipidomics on the plasma of a unique cohort of 73 monozygotic twins discordant for MS. We analyzed 243 lipid species, evaluated lipid features such as fatty acyl chain length and number of acyl chain double bonds, and detected phospholipids that were significantly altered in the plasma of co-twins with MS compared to their non-affected siblings. Strikingly, changes were most prominent in ether phosphatidylethanolamines and ether phosphatidylcholines, suggesting a role for altered lipid signaling in the disease.

Antibody signatures in patients with histopathologically defined multiple sclerosis patterns.

Early active multiple sclerosis (MS) lesions can be classified histologically into three main immunopathological patterns of demyelination (patterns I-III), which suggest pathogenic heterogeneity and may predict therapy response. Patterns I and II show signs of immune-mediated demyelination, but only pattern II is associated with antibody/complement deposition. In pattern III lesions, which include Baló's concentric sclerosis, primary oligodendrocyte damage was proposed. Serum antibody reactivities could reflect disease pathogenesis and thus distinguish histopathologically defined MS patterns. We established a customized microarray with more than 700 peptides that represent human and viral antigens potentially relevant for inflammatory demyelinating CNS diseases, and tested sera from 66 patients (pattern I n = 12; II n = 29; III n = 25, including 8 with Baló's), healthy controls, patients with Sjögren's syndrome and stroke patients. Cell-based assays were performed for aquaporin 1 (AQP1) and AQP4 antibody detection. No single peptide showed differential binding among study cohorts. Because antibodies can react with different peptides from one protein, we also analyzed groups of peptides. Patients with pattern II showed significantly higher reactivities to Nogo-A peptides as compared to patterns I (p = 0.02) and III (p = 0.02). Pattern III patients showed higher reactivities to AQP1 (compared to pattern I p = 0.002, pattern II p = 0.001) and varicella zoster virus (VZV, compared to pattern II p = 0.05). In patients with Baló's, AQP1 reactivity was also significantly higher compared to patients without Baló's (p = 0.04), and the former revealed distinct antibody signatures. Histologically, Baló's patients showed loss of AQP1 and AQP4 in demyelinating lesions, but no antibodies binding conformational AQP1 or AQP4 were detected. In summary, higher reactivities to Nogo-A peptides in pattern II patients could be relevant for enhanced axonal repair and remyelination. Higher reactivities to AQP1 peptides in pattern III patients and its subgroup of Baló's patients possibly reflect astrocytic damage. Finally, latent VZV infection may cause peripheral immune activation.

DNA methylation signatures of monozygotic twins clinically discordant for multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system with a modest concordance rate in monozygotic twins, which strongly argues for involvement of epigenetic factors. We observe highly similar peripheral blood mononuclear cell-based methylomes in 45 MS-discordant monozygotic twins. Nevertheless, we identify seven MS-associated differentially methylated positions (DMPs) of which we validate two, including a region in the TMEM232 promoter and ZBTB16 enhancer. In CD4 + T cells we find an MS-associated differentially methylated region in FIRRE. Additionally, 45 regions show large methylation differences in individual pairs, but they do not clearly associate with MS. Furthermore, we present epigenetic biomarkers for current interferon-beta treatment, and extensive validation shows that the ZBTB16 DMP is a signature for prior glucocorticoid treatment. Taken together, this study represents an important reference for epigenomic MS studies, identifies new candidate epigenetic markers, and highlights treatment effects and genetic background as major confounders.

Mitochondrial DNA Variation and Heteroplasmy in Monozygotic Twins Clinically Discordant for Multiple Sclerosis.

We examined the debated link between mitochondrial DNA (mtDNA) variation and multiple sclerosis (MS) using 49 monozygotic (MZ) twin pairs clinically discordant for MS, which enables to associate de novo mtDNA variants, skewed heteroplasmy, and mtDNA copy number with MS manifestation. Ultra-deep sequencing of blood-derived mtDNA revealed 25 heteroplasmic variants with potentially pathogenic features in 18 pairs. All variants were pair-specific and had low and/or similar heteroplasmy levels in both cotwins. In one pair, a confirmed pathogenic variant (m.11778G>A, heteroplasmy ∼50%) associated with Leber hereditary optic neuropathy was detected. Detailed diagnostic investigation revealed subclinical MS signs in the prior nondiseased cotwin. Moreover, neither mtDNA deletions nor copy-number variations were involved. Furthermore, the majority of heteroplasmic variants were shared among MZ twins and exhibited more similar heteroplasmy levels in the same tissue of MZ twins as compared with different tissues of the same individual. Heteroplasmy levels were also more similar within MZ twins compared with nonidentical siblings. Our analysis excludes mtDNA variation as a major driver of the discordant clinical manifestation of MS in MZ twins, and provides valuable insights into the occurrence and distribution of heteroplasmic variants within MZ twins and nonidentical siblings, and across different tissues.