Nimodipine fosters remyelination in a mouse model of multiple sclerosis and induces microglia-specific apoptosis.

Despite continuous interest in multiple sclerosis (MS) research, there is still a lack of neuroprotective strategies, because the main focus has remained on modulating the immune response. Here we performed in-depth analysis of neurodegeneration in experimental autoimmune encephalomyelitis (EAE) and in in vitro studies regarding the effect of the well-established L-type calcium channel antagonist nimodipine. Nimodipine treatment attenuated clinical EAE and spinal cord degeneration and promoted remyelination. Surprisingly, we observed calcium channel-independent effects on microglia, resulting in apoptosis. These effects were cell-type specific and irrespective of microglia polarization. Apoptosis was accompanied by decreased levels of nitric oxide (NO) and inducible NO synthase (iNOS) in cell culture as well as decreased iNOS and reactive oxygen species levels in EAE. In addition, increased numbers of Olig2+APC+ oligodendrocytes were detected. Overall, nimodipine application seems to generate a favorable environment for regenerative processes and therefore could be a treatment option for MS, because it combines features of immunomodulation with beneficial effects on neuroregeneration.

Nav1.6 promotes inflammation and neuronal degeneration in a mouse model of multiple sclerosis.

BACKGROUND: In multiple sclerosis (MS) and in the experimental autoimmune encephalomyelitis (EAE) model of MS, the Nav1.6 voltage-gated sodium (Nav) channel isoform has been implicated as a primary contributor to axonal degeneration. Following demyelination Nav1.6, which is normally co-localized with the Na+/Ca2+ exchanger (NCX) at the nodes of Ranvier, associates with ß-APP, a marker of neural injury. The persistent influx of sodium through Nav1.6 is believed to reverse the function of NCX, resulting in an increased influx of damaging Ca2+ ions. However, direct evidence for the role of Nav1.6 in axonal degeneration is lacking. METHODS: In mice floxed for Scn8a, the gene that encodes the α subunit of Nav1.6, subjected to EAE we examined the effect of eliminating Nav1.6 from retinal ganglion cells (RGC) in one eye using an AAV vector harboring Cre and GFP, while using the contralateral either injected with AAV vector harboring GFP alone or non-targeted eye as control. RESULTS: In retinas, the expression of Rbpms, a marker for retinal ganglion cells, was found to be inversely correlated to the expression of Scn8a. Furthermore, the gene expression of the pro-inflammatory cytokines Il6 (IL-6) and Ifng (IFN-γ), and of the reactive gliosis marker Gfap (GFAP) were found to be reduced in targeted retinas. Optic nerves from targeted eyes were shown to have reduced macrophage infiltration and improved axonal health. CONCLUSION: Taken together, our results are consistent with Nav1.6 promoting inflammation and contributing to axonal degeneration following demyelination.

Anti-CD52 antibody treatment depletes B cell aggregates in the central nervous system in a mouse model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) for which several new treatment options were recently introduced. Among them is the monoclonal anti-CD52 antibody alemtuzumab that depletes mainly B cells and T cells in the immune periphery. Considering the ongoing controversy about the involvement of B cells and in particular the formation of B cell aggregates in the brains of progressive MS patients, an in-depth understanding of the effects of anti-CD52 antibody treatment on the B cell compartment in the CNS itself is desirable. METHODS: We used myelin basic protein (MBP)-proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 (B6) mice as B cell-dependent model of MS. Mice were treated intraperitoneally either at the peak of EAE or at 60 days after onset with 200 µg murine anti-CD52 vs. IgG2a isotype control antibody for five consecutive days. Disease was subsequently monitored for 10 days. The antigen-specific B cell/antibody response was measured by ELISPOT and ELISA. Effects on CNS infiltration and B cell aggregation were determined by immunohistochemistry. Neurodegeneration was evaluated by Luxol Fast Blue, SMI-32, and Olig2/APC staining as well as by electron microscopy and phosphorylated heavy neurofilament serum ELISA. RESULTS: Treatment with anti-CD52 antibody attenuated EAE only when administered at the peak of disease. While there was no effect on the production of MP4-specific IgG, the treatment almost completely depleted CNS infiltrates and B cell aggregates even when given as late as 60 days after onset. On the ultrastructural level, we observed significantly less axonal damage in the spinal cord and cerebellum in chronic EAE after anti-CD52 treatment. CONCLUSION: Anti-CD52 treatment abrogated B cell infiltration and disrupted existing B cell aggregates in the CNS.

Differential effects of FTY720 on the B cell compartment in a mouse model of multiple sclerosis.

BACKGROUND: MP4-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS), which enables targeted research on B cells, currently much discussed protagonists in MS pathogenesis. Here, we used this model to study the impact of the S1P1 receptor modulator FTY720 (fingolimod) on the autoreactive B cell and antibody response both in the periphery and the central nervous system (CNS). METHODS: MP4-immunized mice were treated orally with FTY720 for 30 days at the peak of disease or 50 days after EAE onset. The subsequent disease course was monitored and the MP4-specific B cell/antibody response was measured by ELISPOT and ELISA. RNA sequencing was performed to determine any effects on B cell-relevant gene expression. S1P1 receptor expression by peripheral T and B cells, B cell subset distribution in the spleen and B cell infiltration into the CNS were studied by flow cytometry. The formation of B cell aggregates and of tertiary lymphoid organs (TLOs) was evaluated by histology and immunohistochemistry. Potential direct effects of FTY720 on B cell aggregation were studied in vitro. RESULTS: FTY720 significantly attenuated clinical EAE when treatment was initiated at the peak of EAE. While there was a significant reduction in the number of T cells in the blood after FTY720 treatment, B cells were only slightly diminished. Yet, there was evidence for the modulation of B cell receptor-mediated signaling upon FTY720 treatment. In addition, we detected a significant increase in the percentage of B220+ B cells in the spleen both in acute and chronic EAE. Whereas acute treatment completely abrogated B cell aggregate formation in the CNS, the numbers of infiltrating B cells and plasma cells were comparable between vehicle- and FTY720-treated mice. In addition, there was no effect on already developed aggregates in chronic EAE. In vitro B cell aggregation assays suggested the absence of a direct effect of FTY720 on B cell aggregation. However, FTY720 impacted the evolution of B cell aggregates into TLOs. CONCLUSIONS: The data suggest differential effects of FTY720 on the B cell compartment in MP4-induced EAE.

The splenic T cell receptor repertoire during an immune response against a complex antigen: Expanding private clones accumulate in the high and low copy number region.

Large cellular antigens comprise a variety of different epitopes leading to a T cell response of extreme diversity. Therefore, tracking such a response by next generation sequencing of the T cell receptor (TCR) in order to identify common TCR properties among the expanding T cells represents an enormous challenge. In the present study we adapted a set of established indices to elucidate alterations in the TCR repertoire regarding sequence similarities between TCRs including VJ segment usage and diversity of nucleotide coding of a single TCR. We combined the usage of these indices with a new systematic splitting strategy regarding the copy number of the extracted clones to divide the repertoire into multiple fractions for separate analysis. We implemented this new analytic approach using the splenic TCR repertoire following immunization with sheep red blood cells (SRBC) in mice. As expected, early after immunization presumably antigen-specific clones accumulated in high copy number fractions, but at later time points similar accumulation of specific clones occurred within the repertoire fractions of lowest copy number. For both repertoire regions immunized animals could reliably be distinguished from control in a classification approach, demonstrating the robustness of the two effects at the individual level. The direction in which the indices shifted after immunization revealed that for both the early and the late effect alterations in repertoire parameters were caused by antigen-specific private clones displacing non-specific public clones. Taken together, tracking antigen-specific clones by their displacement of average TCR repertoire characteristics in standardized repertoire fractions ensures that our analytical approach is fairly independent from the antigen in question and thus allows the in-depth characterization of a variety of immune responses.

Sleep restriction prior to antigen exposure does not alter the T cell receptor repertoire but impairs germinal center formation during a T cell-dependent B cell response in murine spleen.

It is well known that sleep promotes immune functions. In line with this, a variety of studies in animal models and humans have shown that sleep restriction following an antigen challenge dampens the immune response on several levels which leads to e.g. worsening of disease outcome and reduction of vaccination efficiency, respectively. However, the inverse scenario with sleep restriction preceding an antigen challenge is only investigated in a few animal models where it has been shown to reduce antigen uptake and presentation as well as pathogen clearance and survival rates. Here, we use injection of sheep red blood cells to investigate the yet unknown effect on a T cell-dependent B cell response in a well-established mouse model. We found that 6 â€‹h of sleep restriction prior to the antigen challenge does not impact the T cell reaction including the T cell receptor repertoire but dampens the development of germinal centers which correlates with reduced antigen-specific antibody titer indicating an impaired B cell response. These changes concerned a functionally more relevant level than those found in the same experimental model with the inverse scenario when sleep restriction followed the antigen challenge. Taken together, our findings showed that the outcome of the T cell-dependent B cell response is indeed impacted by sleep restriction prior to the antigen challenge which highlights the clinical significance of this scenario and the need for further investigations in humans, for example concerning the effect of sleep restriction preceding a vaccination.

Effects of sleep on the splenic milieu in mice and the T cell receptor repertoire recruited into a T cell dependent B cell response.

Sleep is known to improve immune function ranging from cell distribution in the naïve state to elevated antibody titers after an immune challenge. The underlying mechanisms still remain unclear, partially because most studies have focused on the analysis of blood only. Hence, we investigated the effects of sleep within the spleen in female C57BL/6J mice with normal sleep compared to short-term sleep-deprived animals both in the naïve state and after an antigen challenge. Lack of sleep decreased the expression of genes associated with immune cell recruitment into and antigen presentation within the spleen both in the naïve state and during a T cell dependent B cell response directed against sheep red blood cells (SRBC). However, neither T cell proliferation nor formation of SRBC-specific antibodies was affected. In addition, the T cell receptor repertoire recruited into the immune response within seven days was not influenced by sleep deprivation. Thus, sleep modulated the molecular milieu within the spleen whereas we could not detect corresponding changes in the primary immune response against SRBC. Further studies will show whether sleep influences the secondary immune response against SRBC or the development of the B cell receptor repertoire, and how this can be compared to other antigens.

Danger: High Voltage-The Role of Voltage-Gated Calcium Channels in Central Nervous System Pathology.

Voltage-gated calcium channels (VGCCs) are widely distributed within the central nervous system (CNS) and presumed to play an important role in the pathophysiology of a broad spectrum of CNS disorders including Alzheimer's and Parkinson's disease as well as multiple sclerosis. Several calcium channel blockers have been in clinical practice for many years so that their toxicity and side effects are well studied. However, these drugs are primarily used for the treatment of cardiovascular diseases and most if not all effects on brain functions are secondary to peripheral effects on blood pressure and circulation. While the use of calcium channel antagonists for the treatment of CNS diseases therefore still heavily depends on the development of novel strategies to specifically target different channels and channel subunits, this review is meant to provide an impulse to further emphasize the importance of future research towards this goal.