Browsing by Subject "single-cell RNA sequencing"

Multiple Sclerosis (MS) is an incurable autoimmune demyelinating disease affecting the central nervous system (CNS). Although the detailed pathogenesis remains unclear, recent research has highlighted the involvement of B cells. For decades, however, MS research was based on T cell-focused animal models of autoimmune encephalomyelitis (EAE), which do not reflect the involvement of B cells in the pathogenesis. Therefore, B cell-dependent EAE models are hypothesized to allow a better understanding of MS immunohistopathology and may therefore lead to the development of efficient treatments. In our spontaneous relapsing-remitting (RR) EAE model, B cells are recruited from the endogenous repertoire by transgenic myelin oligodendrocyte glycoprotein (MOG) -reactive T cells, causing the development of EAE in 3–4-month-old mice. Interestingly, MOG-specific antibodies are present long before actual onset of clinical disease and can be detected already in 5-week-old RR mice and disease development in RR mice is dependent both on the presence of (presumably MOG-specific) B cells as well as on stimuli provided by intestinal microbiota. Firstly, we evaluated the broader usability of induced germinal center cell (iGB) culture as a model for B cell repertoire studies. Then, by using iGB culture, we studied whether MOG-specific B cells are present in secondary lymphoid organs of younger than 4-week-old and germ-free RR mice. Finally, this study aimed to investigate whether the repertoire of MOG-specific B cells undergoes significant qualitative changes from young healthy mice to older acutely sick RR mice, and whether at the time of disease onset the recruited MOG-specific B cells expand and mature in the cervical nodes (cLN) or in the CNS. To do so, following the hosting-lab’s previous single-cell RNA sequencing (scRNA-seq) of B cells derived from cLN of 5-week-old RR mice, we performed the scRNA-seq of B cells from CNS, spleen, and cLN of acutely sick RR EAE mice. We demonstrated that iGB culture is an unsuitable tool to expand pre-activated B cells, and hence, in our hands it was inappropriate for repertoire studies. However, iGB culture proved to be useful for screening different organs for MOG-specific B lymphocytes, and we found that anti-MOG antibodies were firstly detected in 3-4-week-old RR mice, and MOG-specific B cells were present also in germ-free RR mice. Our scRNA-seq results revealed many highly expanded MOG-specific B cell clonotypes in acutely sick RR mice. Moreover, the B cell repertoire of sick RR mice was more diverse, including IgG1, IgM, IgG2b, IgG2c, and IgG3 isotypes, compared to healthy 5-week-old RR mice that had only IgG1 or IgM isotypes. Two-thirds of the expanded clonotypes were primarily detected in the CNS in sick RR mice, indicating that clonotypes develop further and continue isotype switching within the CNS. We also detected more somatic mutation in the variable region of expanded clones of sick RR mice compared to 5-week-old RR mice. The results of this study clearly show an antigen-driven evolution of the MOG-specific B cell repertoire from healthy young to acutely sick RR mice, which seems to occur mainly in CNS itself. In contrast, cLN are the major initial priming site of MOG-specific B cells in healthy RR mice, even under germ-free conditions. This suggests that commensal microbiota is not required for initial recruitment of MOG-specific B cells, but for the development of EAE. To further validate our encouraging scRNA-Seq results, it is necessary, in future experiments, to confirm the MOG-specificity of expanded clonotypes.

Cardiovascular diseases are the leading cause of death globally. Especially pathological cardiac hypertrophy can be a trigger for severe pathological conditions, such as congestive heart failure. Previously, overexpression of vascular endothelial growth factor B (VEGF-B) in cardiomyocytes has been shown to lead to cardiac hypertrophy, but in a reversible, physiological way. Furthermore, VEGF-B overexpression leads to significant expansion of the coronary vascular tree. This study compares transcriptomics of postnatal and adult murine cardiac endothelial cells (ECs) and examines the transcriptional changes in response to VEGF-B transgene, plus the effect of the VEGF-B transgene on recovery of the murine cardiac ECs from myocardial infarction (MI). I analyzed isolated ECs from VEGF-B transgenic and AAV-VEGF-B transduced mice with single-cell RNA sequencing. The markers used for identification of the cell types applies to all experimental groups, although the proportions of cells differ among the conditions. The myocardial VEGF-B transgene promotes EC proliferation during development and boosts endothelial proliferation also in adult mice both in physiological conditions and after MI. Trajectory analysis indicates that ECs from the VEGF-B treated mice follow a distinct trajectory to enter the cell cycle after MI. These results suggest VEGF-B gene therapy as a new tool for coronary vessel remodeling, which could open new perspectives in the prevention and treatment of myocardial infarction.

Multiple sclerosis (MS) is one of the most common reasons for neurological disability in young adults, yet the aetiology of the disease remains to be discovered. MS involves an autoimmune reaction in the central nervous system, which results in demyelination, axonal degradation, and inflammation. These result in various symptoms, such as motor and sensory disturbances, cognitive symptoms, fatigue, and problems with balance. MS is chronic and progressive, and medications are used to slow the neuronal damage and reduce relapses. The most evident risk factor for MS is Epstein-Barr virus (EBV) infection, as nearly 100% of MS patients are seropositive for the virus. However, the mechanism how EBV contributes to the disease is not known. A highly sensitive quantitative multiplex PCR method was used to examine reactivation of EBV and eight other human herpesviruses in the saliva of MS patients (n=9) and healthy controls (n=7). Single-cell RNA sequencing methods were used to study the cell composition and expression patterns of cerebrospinal fluid (CSF) in treatment-naïve MS patients at the diagnostic phase (n=4) and in controls (n=4). EBV was found to be shedding in eight out of nine MS patients and in only one control, and the viral load was significantly higher in MS patients. Single-cell sequencing of the CSF revealed that MS induces expansion of antibody producing and cytotoxic cell types. Differential expression analysis found that MS CSF B cells significantly express EBNA1BP2, which plays a crucial role in the replication and partitioning of EBV episomes in infected cells. These results support the involvement of EBV in MS. Better knowledge of the viral role in the onset of MS will be useful in the development potential antiviral drugs and EBV vaccination that could even prevent the disease.