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Myelinmutationen des zentralen und peripheren Nervensystems verursachen erheblich behindernde und bislang nicht heilbare Erkrankungen. In dieser Arbeit verwendeten wir transgene PLP überexprimierende Mäuse (PLPtg) als Modell für zentrale Myelinopathien und heterozygot P0 defiziente (P0+/-) Mäuse als Modell für hereditäre Neuropathien des peripheren Nervensystems. Beide Modelle zeigen eine niedriggradige Inflammation des Nervengewebes. Durch Verpaarung mit immundefizienten Mausstämmen konnten wir die Relevanz von Makrophagen und T- Lymphozyten in der Entstehung der Myelinpathologie zeigen. Nachdem wir beweisen konnten, dass CD8+ T- Lymphozyten maßgeblich zur Pathologie in PLPtg Mäusen beitragen untersuchten wir den Einfluss eines wichtigen zytotoxischen Moleküls, Granzym B, auf den neuralen Schaden. Durch Generierung von Granzym B defizienten PLPtg Knochenmarkschimären konnten wir eine deutliche Reduktion des glialen Schadens und der Oligodendrozytenapoptose nachweisen. Granzym B ist also zumindest teilweise verantwortlich für die Schädigung, die durch T- Lymphozyten hervorgerufen wird. Um die zusätzliche Informationen über die Rolle der Immunmodulation in unseren Modellen zu gewinnen, untersuchten wir das koinhibitorische Molekül PD-1, einen CD-28 verwandten Rezeptor, der auf B- und T- Lymphozyten exprimiert wird. Bei der Untersuchung von Myelinmutanten des ZNS und PNS (PLPtg und P0+/-), die zusätzlich PD-1 defizient waren, konnten wir einen signifikanten Anstieg von CD8+ T- Lymphozyten und eine deutliche Verschlechterung des glialen Schadens beobachten. In PLPtg Mäusen induzierte die Abwesenheit von PD-1 verstärkte Oligodendrozytenapoptose und klonale Expansion. Außerdem neigen ZNS- Lymphozyten aber nicht periphere CD8+ T- Zellen zur verstärkten Sekretion von proinflammatorischen Zytokinen. In P0+/- Mäusen führt Abwesenheit von PD-1 zu moderaten motorischen und sensorischen Störungen, was die wichtige Rolle von PD-1 in immunologischen Regulationsmechanismen unterstreicht. Zusammenfassend kann man festhalten, daß Granzym B ein wichtiges Effektormolekül zytotoxischer T- Zellen in PLPtg Mäusen ist. PD-1 spielt eine wichtige Rolle in der Regulation von Effektorzellen in unseren Modellen für zentrale und periphere Myelinopathien. Veränderungen dieser Regulation können deutliche Neuroinflammation mit starker Myelinpathologie hervorrufen. Diese Ergebnisse können dazu beitragen, die starke klinische Variabilität von polygenen und sogar monogenen neurologischen Erkrankungen zu erklären.
Background: The diagnostic and pathophysiological relevance of antibodies to aquaporin-4 (AQP4-Ab) in patients with neuromyelitis optica spectrum disorders (NMOSD) has been intensively studied. However, little is known so far about the clinical impact of AQP4-Ab seropositivity.
Objective: To analyse systematically the clinical and paraclinical features associated with NMO spectrum disorders in Caucasians in a stratified fashion according to the patients' AQP4-Ab serostatus.
Methods: Retrospective study of 175 Caucasian patients (AQP4-Ab positive in 78.3%).
Results: Seropositive patients were found to be predominantly female (p < 0.0003), to more often have signs of co-existing autoimmunity (p < 0.00001), and to experience more severe clinical attacks. A visual acuity of <= 0.1 during acute optic neuritis (ON) attacks was more frequent among seropositives (p < 0.002). Similarly, motor symptoms were more common in seropositive patients, the median Medical Research Council scale (MRC) grade worse, and MRC grades <= 2 more frequent, in particular if patients met the 2006 revised criteria (p < 0.005, p < 0.006 and p < 0.01, respectively), the total spinal cord lesion load was higher (p < 0.006), and lesions >= 6 vertebral segments as well as entire spinal cord involvement more frequent (p < 0.003 and p < 0.043). By contrast, bilateral ON at onset was more common in seronegatives (p < 0.007), as was simultaneous ON and myelitis (p < 0.001); accordingly, the time to diagnosis of NMO was shorter in the seronegative group (p < 0.029). The course of disease was more often monophasic in seronegatives (p < 0.008). Seropositives and seronegatives did not differ significantly with regard to age at onset, time to relapse, annualized relapse rates, outcome from relapse (complete, partial, no recovery), annualized EDSS increase, mortality rate, supratentorial brain lesions, brainstem lesions, history of carcinoma, frequency of preceding infections, oligoclonal bands, or CSF pleocytosis. Both the time to relapse and the time to diagnosis was longer if the disease started with ON (p < 0.002 and p < 0.013). Motor symptoms or tetraparesis at first myelitis and > 1 myelitis attacks in the first year were identified as possible predictors of a worse outcome.
Mice overexpressing proteolipid protein (PLP) develop a leukodystrophy-like disease involving cytotoxic, CD8+ T-lymphocytes. Here we show that these cytotoxic T-lymphocytes perturb retrograde axonal transport. Using fluorogold stereotactically injected into the colliculus superior, we found that PLP overexpression in oligodendrocytes led to significantly reduced retrograde axonal transport in retina ganglion cell axons. We also observed an accumulation of mitochondria in the juxtaparanodal axonal swellings, indicative for a disturbed axonal transport. PLP overexpression in the absence of T-lymphocytes rescued retrograde axonal transport defects and abolished axonal swellings. Bone marrow transfer from wildtype mice, but not from perforin- or granzyme B-deficient mutants, into lymphocyte-deficient PLP mutant mice led again to impaired axonal transport and the formation of axonal swellings, which are predominantly located at the juxtaparanodal region. This demonstrates that the adaptive immune system, including cytotoxic T-lymphocytes which release perforin and granzyme B, are necessary to perturb axonal integrity in the PLP-transgenic disease model. Based on our observations, so far not attended molecular and cellular players belonging to the immune system should be considered to understand pathogenesis in inherited myelin disorders with progressive axonal damage.
Dimethyl fumarate (DMF) is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS) by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF) modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 \(\mu\)M blocked the IL-1\(\beta\)-induced nuclear translocation of NF-\(\kappa\)B/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1\(\beta\)-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1\(\beta\)-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells.
Background: Despite pleiotropic immunomodulatory effects of apolipoprotein E (apoE) in vitro, its effects on the clinical course of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) are still controversial. As sex hormones modify immunomodulatory apoE functions, they may explain contentious findings. This study aimed to investigate sex-specific effects of apoE on disease course of EAE and MS.
Methods: MOG\(_{35-55}\) induced EAE in female and male apoE-deficient mice was assessed clinically and histopathologically. apoE expression was investigated by qPCR. The association of the MS severity score (MSSS) and APOE rs429358 and rs7412 was assessed across 3237 MS patients using linear regression analyses.
Results: EAE disease course was slightly attenuated in male apoE-deficient (apoE\(^{-/-}\)) mice compared to wildtype mice (cumulative median score: apoE\(^{-/-}\) = 2 [IQR 0.0-4.5]; wildtype = 4 [IQR 1.0-5.0]; n = 10 each group, p = 0.0002). In contrast, EAE was more severe in female apoE\(^{-/-}\) mice compared to wildtype mice (cumulative median score: apoE\(^{-/-}\) = 3 [IQR 2.0-4.5]; wildtype = 3 [IQR 0.0-4.0]; n = 10, p = 0.003). In wildtype animals, apoE expression during the chronic EAE phase was increased in both females and males (in comparison to naive animals; p < 0.001). However, in MS, we did not observe a significant association between MSSS and rs429358 or rs7412, neither in the overall analyses nor upon stratification for sex.
Conclusions: apoE exerts moderate sex-specific effects on EAE severity. However, the results in the apoE knock-out model are not comparable to effects of polymorphic variants in the human APOE gene, thus pinpointing the challenge of translating findings from the EAE model to the human disease.
NKG2D is an activating receptor on T cells, which has been implicated in the pathogenesis of autoimmune diseases. T cells are critically involved in idiopathic inflammatory myopathies (IIM) and have been proposed as specific therapeutic targets. However, the mechanisms underlying T cell-mediated progressive muscle destruction in IIM remain to be elucidated. We here determined the involvement of the NKG2D - IL-15 signaling pathway. Primary human myoblasts expressed NKG2D ligands, which were further upregulated upon inflammatory stimuli. In parallel, shedding of the soluble NKG2D ligand MICA (sMICA) decreased upon inflammation potentially diminishing inhibition of NKG2D signaling. Membrane-related expression of IL-15 by myoblasts induced differentiation of naive CD8\(^+\) T cells into highly activated, cytotoxic \(CD8^+NKG2D^{high}\) T cells demonstrating NKG2D-dependent lysis of myoblasts in vitro. \(CD8^+NKG2D^{high}\) T cell frequencies were increased in the peripheral blood of polymyositis (PM) patients and correlated with serum creatinine kinase concentrations, while serum sMICA levels were not significantly changed. In muscle biopsy specimens from PM patients expression of the NKG2D ligand MICA/B was upregulated, IL-15 was expressed by muscle cells, CD68\(^+\) macrophages as well as CD4\(^+\) T cells, and \(CD8^+NKG2D^+\) cells were frequently detected within inflammatory infiltrates arguing for a local signaling circuit in the inflammatory muscle milieu. In conclusion, the NKG2D - IL-15 signaling pathway contributes to progressive muscle destruction in IIM potentially opening new therapeutic avenues.
B cells have only recently begun to attract attention in the immunopathology of multiple sclerosis (MS). Suitable markers for the prediction of treatment success with immunomodulatory drugs are still missing. Here we evaluated the B cell response to brain antigens in n = 34 relapsing-remitting MS (RRMS) patients treated with glatiramer acetate (GA) using the enzyme-linked immunospot technique (ELISPOT). Our data demonstrate that patients can be subdivided into responders that show brain-specific B cell reactivity in the blood and patients without this reactivity. Only in patients that classified as B cell responders, there was a significant positive correlation between treatment duration and the time since last relapse in our study. This correlation was GA-specific because it was absent in a control group that consisted of interferon-\(\beta\) (IFN-\(\beta\))-treated RRMS patients (n = 23). These data suggest that GA has an effect on brain-reactive B cells in a subset of patients and that only this subset benefits from treatment. The detection of brain-reactive B cells is likely to be a suitable tool to identify drug responders.
Lymphocytes express potassium channels that regulate physiological cell functions, such as activation, proliferation and migration. Expression levels of K\(_{2P}\)5.1(TASK2; KCNK5) channels belonging to the family of two-pore domain potassium channels have previously been correlated to the activity of autoreactive T lymphocytes in patients with multiple sclerosis and rheumatoid arthritis. In humans, K\(_{2P}\)5.1 channels are upregulated upon T cell stimulation and influence T cell effector functions. However, a further clinical translation of targeting K\(_{2P}\)5.1 is currently hampered by a lack of highly selective inhibitors, making it necessary to evaluate the impact of KCNK5 in established preclinical animal disease models. We here demonstrate that K\(_{2P}\)5.1 knockout (K\(_{2P}\)5.1\(^{-/-}\) mice display no significant alterations concerning T cell cytokine production, proliferation rates, surface marker molecules or signaling pathways. In an experimental model of autoimmune neuroinflammation, K\(_{2P}\)5.1\(^{-/-}\) mice show a comparable disease course to wild-type animals and no major changes in the peripheral immune system or CNS compartment. A compensatory upregulation of the potassium channels K\(_{2P}\)3.1 and K\(_{V}\)1.3 seems to counterbalance the deletion of K\(_{2P}\)5.1. As an alternative model mimicking autoimmune neuroinflammation, experimental autoimmune encephalomyelitis in the common marmoset has been proposed, especially for testing the efficacy of new potential drugs. Initial experiments show that K\(_{2P}\)5.1 is functionally expressed on marmoset T lymphocytes, opening up the possibility for assessing future K\(_{2P}\)5.1-targeting drugs.
Multiple sclerosis (MS) is a prevalent neurological disease of complex etiology. Here, we describe the characterization of a multi-incident MS family that nominated a rare missense variant (p.G420D) in plasminogen (PLG) as a putative genetic risk factor for MS. Genotyping of PLG p.G420D (rs139071351) in 2160 MS patients, and 886 controls from Canada, identified 10 additional probands, two sporadic patients and one control with the variant. Segregation in families harboring the rs139071351 variant, identified p.G420D in 26 out of 30 family members diagnosed with MS, 14 unaffected parents, and 12 out of 30 family members not diagnosed with disease. Despite considerably reduced penetrance, linkage analysis supports cosegregation of PLG p.G420D and disease. Genotyping of PLG p.G420D in 14446 patients, and 8797 controls from Canada, France, Spain, Germany, Belgium, and Austria failed to identify significant association with disease (P = 0.117), despite an overall higher prevalence in patients (OR = 1.32; 95% CI = 0.93–1.87). To assess whether additional rare variants have an effect on MS risk, we sequenced PLG in 293 probands, and genotyped all rare variants in cases and controls. This analysis identified nine rare missense variants, and although three of them were exclusively observed in MS patients, segregation does not support pathogenicity. PLG is a plausible biological candidate for MS owing to its involvement in immune system response, blood-brain barrier permeability, and myelin degradation. Moreover, components of its activation cascade have been shown to present increased activity or expression in MS patients compared to controls; further studies are needed to clarify whether PLG is involved in MS susceptibility.
Differential effects of FTY720 on the B cell compartment in a mouse model of multiple sclerosis.
(2017)
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. S1P\(_{1}\) 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.