Impact of B cell secreted factors on myelin in progressive multiple sclerosis
Dabrowska, Malgorzata Natalia
Dabrowska, Malgorzata Natalia
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Identifiers
https://hdl.handle.net/10379/18602
https://doi.org/10.13025/29396
https://doi.org/10.13025/29396
Repository DOI
Publication Date
2025-02-24
Type
doctoral thesis
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Abstract
Ex vivo organotypic slice models present several advantages over conventional cell culture systems by enabling the investigation of multiple cell types within their native 3D architecture, while preserving intrinsic cell-cell interactions and signalling pathways. In approximately 40% of individuals with progressive multiple sclerosis (PMS), B cell niches, referred to as ectopic lymphoid follicles (ELF), are observed in cortical sulci and are associated with cortical demyelination. This PhD thesis focused on developing a novel hybrid human-rat ex vivo platform to assess the cytotoxicity of B cells from individuals with PMS, hypothesizing that co-culture with brain slices may induce PMS-like cortical demyelination.
Coronal organotypic brain slice (OBS) cultures were optimized using both neonatal and adult rat brains. Initially, evaluation of the metabolic activity of slice cultures from neonatal rats (P10/11) was investigated. The data indicated that caudal slices (Bregma -1.10mm to -3.50mm) exhibited 29.6% higher metabolic activity than rostral slices (p<0.001, n=50). Furthermore, cortical slices without structural connections exhibited 38.5% lower metabolic activity than those with intact connections (p<0.001, n=26). Severing these connections resulted in a 25.5% reduction in metabolic activity (p<0.02, n=20), emphasizing the necessity of maintaining anatomical integrity and connectivity for ex vivo cortical studies. The caudal brain regions provided optimal metabolic activity and extended viability, making them ideal for mimicking cortical physiology under healthy and diseased conditions.
Adult rat brain slices typically exhibit limited viability in ex vivo cultures (often not exceeding 7 days, so fetal bovine serum (FBS) concentrations (0-10%) in growth media were tested. Cortical slices cultured in 10% FBS demonstrated significantly higher metabolic activity (p<0.001, N=4) compared to those cultured with reduced FBS concentrations, prolonging viability to 21 days. Caudal slices (~Bregma -1.33mm) remained more metabolically active (p<0.05) than rostral slices.
Since hybrid human-rat models, using LPC to induce demyelination, were being developed, culture conditions compatible with both neonatal rat OBS and human B cells had to be optimised. In the absence of FBS, slices treated with 0.5mg/ml LPC exhibited an 88% reduction in metabolic activity at T2 (p<0.001), and a 67% reduction at T4 (p<0.01) compared to untreated controls. The addition of 10% FBS improved metabolic activity, reducing cytotoxicity at T2 and T4, but tissue integrity was lost. The optimal FBS concentration for
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culturing both neonatal OBS and B cells was found to be 2%, which preserved tissue integrity and prevented cytotoxic effects.
Coronal OBS cultures were then treated with B cell supernatant, from cultures of peripheral blood from PMS+ and PMS- individuals. Proteome Profiler Rat XL Cytokine Array analyses demonstrated that OBS cultures treated with PMS+ supernatant exhibited cytokine expression patterns consistent with clinical PMS, including upregulation of IL-1, IL-17, and IL-22. PMS+ samples also showed the highest cytotoxicity at T2 (p<0.001, N=5) and significantly reduced myelin thickness (quantified on Image J using anti-MOG staining) at T2 (p<0.05) and T4 (p<0.01), disappearing by day T7 (N=5, n= 360). These findings suggest that factors released by PMS+ B cells are cytotoxic to myelin, inducing cortical demyelination in the OBS model.
Next, a series of pilot studies were carried out to further examine the potential of rodent models of MS. Firstly, a study of activated mouse B cell migration in the brain demonstrated that a cuprizone-induced inflammatory environment promotes B cell retention in brain sections, particularly when treated with chemokine ligand 13 (CXCL13), compared to LPC or untreated controls (~30.6% and ~57.4% cells less, respectively). This highlights the necessity of developing an ex vivo model of PMS-like pathology and reinforces the role of B cells in MS-associated cortical myelin toxicity. Injecting B cells into an inflamed brain milieu facilitates their retention despite active glymphatic and cerebrospinal fluid flow. Secondly, further understanding of an inflamed brain milieu was sought by experimenting with biomaterials that could potentially be used to model ELF-like structures. For that purpose, hydrogel (HG) formulations were tested to obtain the storage modulus most closely resembling the malignant human lymph node, as that of ELFs has not yet been documented, and a non-adherent cell line (THP1) was successfully embedded within the hydrogel's 3D structure, showing proof-of-concept. Thirdly, attempts were made to mimic ELFs by promoting extracellular matrix (ECM) deposition in meningeal cultures, using the macromolecular crowder (MMC) carrageenan (CG). Unfortunately, at higher concentrations, cells treated with CG exhibited significant cell death, and no acceleration of collagen deposition was observed.
Finally, the established ex vivo model was used to test the effect on remyelination of a peptide previously shown to be immunomodulatory in vivo. Peptide FhHDM, and its truncated version, were tested on demyelinated cerebellar slices (0.5mg/ml LPC). Treatment with full-length FhHDM resulted in improved metabolic activity and reduced cytotoxicity, suggesting a
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protective effect on myelin. However, further tests and quantification of the myelin area are required to confirm the peptide’s myelin-rescuing potential.
Overall, this model has been extensively optimised and tested and introduces a novel platform for using adult OBS cultures to study neurodegenerative diseases, including drug screening and cortical pathology analysis, particularly in conditions like MS.
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Publisher
University of Galway
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Attribution-NonCommercial-NoDerivatives 4.0 International