Author Of 2 Presentations
FC03.03 - Depicting multiple sclerosis pathology at 160μm isotropic resolution by human whole-brain postmortem 3T magnetic resonance imaging
Abstract
Background
Postmortem magnetic resonance imaging (MRI) of formalin-fixed healthy and diseased human brains with ultra-high spatial resolution has the great potential to depict tissue architecture in fine detail, allowing a deeper understanding of pathological processes. Whole-brain imaging is important since it provides neuroanatomic relationships, reference points across distant brain regions, and a comprehensive view of pathologies affecting the brain. However, ultra-high-resolution whole-brain postmortem MRI is challenging and has been so far almost exclusively performed at 7T with specialized hardware.
Objectives
To develop a 3D isotropic 160µm ultra-high-resolution imaging (URI) approach for human whole-brain ex vivo acquisitions on a standard clinical 3T MRI system. To explore the sensitivity and specificity of the approach to specific pathological features of multiple sclerosis (MS).
Methods
A fixed whole human brain from a patient with secondary progressive MS was investigated. Acquisitions were performed on a clinical 3T Siemens Prismafit MRI system with standard hardware components. URI is based on a gradient echo sequence similar to the 7T approach by Edlow et al. 2019. However, it allows to acquire an isotropic 160µm resolution with low hardware demands and to directly reconstruct the image data on the standard 3T MRI system. URI images display a strong, susceptibility-enhanced tissue contrast.
Results
The reconstructed URI images depicted with remarkable quality the diseased human MS brain at 3T field strength. URI allowed to distinguish fine anatomical details such as the subpial molecular layer, the stria of Gennari as well as some intrathalamic nuclei. Additionally, because of the unprecedented spatial resolution and contrast at 3T, URI permitted to easily identify the presence of subpial lesions, detailed features of intracortical lesions such the presence of incomplete/complete iron rims or patterns of iron accumulation in the entire lesion core in both cortical and white matter lesions (CLs/WMLs), lesions affecting the convoluted layers of the cerebellar cortex and nascent submillimetric CLs/WMLs.
Conclusions
URI provides a comprehensive microscopic insight into the whole-human brain at 3T through the micrometric resolution and a tissue-specific, susceptibility-enhanced contrast. We propose URI as an excellent approach to investigate microscopic brain changes of complex pathologies like MS.
PS06.05 - B cells regulate chronic CNS inflammation in an IL-10-dependent manner
Abstract
Background
Several lines of evidence indicate essential roles for B cells in the pathogenesis of multiple sclerosis (MS). B cells act as potent antigen-presenting cells and throughout the chronic course of MS, B cell-follicle like structures can be found in the meninges of MS patients. However, whether and how B cells interact with CNS-resident cells, such as microglia and astrocytes to possibly modulate chronic progression of MS remains unclear.
Objectives
In the present study, we aimed at analyzing the interaction of B cells with CNS-resident cells in modulation of chronic CNS inflammation.
Methods
Primary microglia and astrocytes were generated from newborn C57BL/6 mice and were incubated with activated B cells or their supernatants. IL-6 and IL-10 production was abolished by genetic ablation or neutralization of IL-6 or IL-10 using specific antibodies. Thereafter, CNS resident cells were co-cultured with MOG-specific T cells. Further, C57BL/6 mice were depleted of B cells by 3 weekly subcutaneous injections of 0.2 mg murine anti-CD20 prior to immunization with MOG peptide p35-55, a setting in which B cells remain naïve. Microglial and astrocytic activation/modulation was assessed by ELISA, flow cytometry, immunohistochemistry and qRT-PCR.
Results
Incubation of primary microglia or astrocytes with IL-10-neutralized B cell supernatant or co-culture with IL-10-deficient B cells resulted in increased pro-inflammatory cytokine production, an upregulation of co-stimulatory molecules as well as an enhanced capacity to activate T cells as antigen-presenting cells. In vivo, depletion of naïve B cells worsened clinical severity of experimental encephalomyelitis (EAE) and increased the number of CNS infiltrating immune cells. Exacerbation was associated with an enhanced expression of molecules involved in antigen-presentation on microglia cells as well as an upregulation of pro-inflammatory gene products in astrocytes.
Conclusions
These findings highlight that B cells substantially alter the functional status of microglia and astrocytes in chronic CNS inflammation. Specifically, B cell-derived IL-10 is capable of diminishing the inflammatory responses of CNS-resident microglia and astrocytes. Our observation suggests that regulatory B cell function may be important in controlling CNS intrinsic inflammation associated with clinical progression.
Author Of 2 Presentations
P0334 - Evobrutinib, a highly selective BTK inhibitor, prevents antigen-activation of B cells and ameliorates experimental autoimmune encephalomyelitis (ID 1125)
Abstract
Background
Background: B cells are key mediators of inflammatory processes in multiple sclerosis, a notion substantiated by the success of B-cell depletion therapies; however, overall depletion does not only target pathogenic B cells but can also affect regulatory B-cell properties. An alternative strategy may be the specific inhibition of Bruton’s tyrosine kinase (BTK), which is centrally involved in B-cell receptor (BCR) signaling and subsequently mediates B-cell activation and differentiation. BTK inhibitors therefore hold the promise to control pathogenic functions such as antigen presentation and cytokine release.
Objectives
Objectives: To evaluate the BTK inhibitor evobrutinib in a mouse model of experimental autoimmune encephalomyelitis (EAE).
Methods
Methods: C57Bl/6 mice received oral evobrutinib or vehicle starting 7 days before immunization with conformational MOG1-117 protein (a B cell–mediated model of EAE). EAE severity was assessed for 60 days using a standard scale. B-cell maturation and activation markers on B and T cells were analyzed by flow cytometry on day 12 post immunization. T cell proliferation and differentiation were assessed after a 3-day co-culture with BTKi-treated B cells. Intracellular calcium flux was analyzed using calcium-sensitive dyes and BCR or T cell receptor (TCR) stimulation. BTK expression and phosphorylation as well as cytokine production were assessed on healthy human B cells via PhosFlow protocols or ELISA, respectively.
Results
Results: Evobrutinib showed a dose-dependent amelioration of EAE severity throughout the 60-day observation period. Evobrutinib led to an accumulation of follicular type (FO) II B cells and a corresponding reduction in FO I B cells, a BTK-dependent transition. Expression of CD86, CD69, and major histocompatibility complex class II on B cells, and CD25 and CD69 on T cells, was reduced. Evobrutinib inhibited the B cell-mediated proliferation and proinflammatory differentiation of T cells. BCR-mediated mobilization of excitatory calcium was reduced by evobrutinib, while TCR signaling remained unaffected. In human B cells, BTK expression and phosphorylation were depending on the maturation of B cells, while the overall cytokine release was inhibited by evobrutinib.
Conclusions
Conclusion: Evobrutinib efficiently reduces BTK-dependent signaling after BCR stimulation, preventing B-cell activation, proinflammatory differentiation, and function. This translates into reduced CNS inflammation and clinical amelioration in a B cell–mediated EAE model.
P0624 - Quantitative multiparametric 3T-MRI of postmortem multiple sclerosis whole brains (ID 1583)
Abstract
Background
Postmortem MRI provides precious insights into the relation of MRI metrics to pathoanatomical features of multiple sclerosis (MS) and can help to understand the basis of damage and repair.
Objectives
To investigate the respective features of MS lesions in the cortex and in the white matter using multiparametric postmortem MR imaging at 3T and identify discriminant characteristics of white matter lesion subgroups.
Methods
We scanned three fixed brains of secondary-progressive MS patients (mean disease duration 15.3 years) on a standard clinical 3T-MRI scanner with following sequences: Magnetization Transfer Saturation (MTsat), T1-relaxometry (T1-rt), Myelin Water Fraction (MWF) and Diffusion Tensor - Fractional Anisotropy (DTI-FA). We compared these metrics between (i) cortical lesions (CL, n=118) and normal-appearing grey matter (NAGM, n=186) and (ii) white matter lesions (WML, n=140) and normal-appearing white matter (NAWM, n=53) using a Mann-Whitney U test. Then, we analyzed the differences between different subgroups of WML (periventricular lesions -PVL-, n=38, WM part of leukocortical lesions -WMLCL-, n=36, subcortical lesions -SCL-, n=66, and areas of “dirty white matter” -DWM-, n=15) by performing a Kruskal-Wallis test and a Mann-Whitney U tests for direct comparison. Bonferroni correction for multiple-testing was applied.
Results
CL exhibited lower MTsat (p<0.001), higher T1-rt (p<0.001) and MWF (p<0.01) than normal appearing cortical tissue. WML showed lower MTsat (p<0.001), higher T1-rt (p<0.001), and lower MWF (p<0.001) than normal appearing white matter. DTI-FA did not differ between CL/WML and NAWM/NAGM. MTsat values were lower in the PVL (p<0.001) and higher in the DWM (p<0.001) in comparison to all other lesion subgroups. T1-rt were higher in PVL (p<0.001) compared to the other lesion subgroups. MWF values were higher in DWM and SCL (p<0.01), not statistically different between PVL and WMLCL. DTI-FA values were lower in WMLCL in comparison to all other subgroups (p<0.01) and did not differ between the other categories.
Conclusions
Postmortem MRI metrics in WML/CL as well as in different subgroups of WML, are compatible with myelin damage and tissue destruction. Interestingly, MWF was higher in CL than in NAGM, which might correspond to a predominance of “myelin blistering” pathology in the cortex. Ongoing work aims to directly correlate our findings with detailed histopathological characterization including electron microscopy of myelin damage.