Neuroinflammation by cytotoxic T-lymphocytes impairs retrograde axonal transport in an oligodendrocyte mutant mouse

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

PD-1 Regulates Neural Damage in Oligodendroglia-Induced Inflammation

We investigated the impact of immune regulatory mechanisms involved in the modulation of the recently presented, CD8+ lymphocyte mediated immune response in a mouse model of oligodendropathy-induced inflammation (PLPtg-mutants). The focus was on the role of the co-inhibitory molecule PD-1, a CD28-related receptor expressed on activated T- and B-lymphocytes associated with immune homeostasis and autoimmunity. PLPtg/PD-1-deficient double mutants and the corresponding bone marrow chimeras were generated and analysed using immunohistochemistry, light- and electron microscopy, with particular emphasis on immune-cell number and neural damage. In addition, the immune cells in both the CNS and the peripheral immune system were investigated by IFN-gamma elispot assays and spectratype analysis. We found that mice with combined pathology exhibited significantly increased numbers of CD4+ and CD8+ T-lymphocytes in the CNS. Lack of PD-1 substantially aggravated the pathological phenotype of the PLPtg mutants compared to genuine PLPtg mutants, whereas the PD-1 deletion alone did not cause alterations in the CNS. CNS T-lymphocytes in PLPtg/PD-1-/- double mutants exhibited massive clonal expansions. Furthermore, PD-1 deficiency was associated with a significantly higher propensity of CNS but not peripheral CD8+ T-cells to secrete proinflammatory cytokines. PD-1 could be identified as a crucial player of tissue homeostasis and immune-mediated damage in a model of oligodendropathy-induced inflammation. Alterations of this regulatory pathway lead to overt neuroinflammation of high pathogenetic impact. Our finding may have implications for understanding the mechanisms leading to the high clinical variability of polygenic or even monogenic disorders of the nervous system

Genome-wide significant association with seven novel multiple sclerosis risk loci

Objective: A recent large-scale study in multiple sclerosis (MS) using the ImmunoChip platform reported on 11 loci that showed suggestive genetic association with MS. Additional data in sufficiently sized and independent data sets are needed to assess whether these loci represent genuine MS risk factors. Methods: The lead SNPs of all 11 loci were genotyped in 10 796 MS cases and 10 793 controls from Germany, Spain, France, the Netherlands, Austria and Russia, that were independent from the previously reported cohorts. Association analyses were performed using logistic regression based on an additive model. Summary effect size estimates were calculated using fixed-effect meta-analysis. Results: Seven of the 11 tested SNPs showed significant association with MS susceptibility in the 21 589 individuals analysed here. Meta-analysis across our and previously published MS case-control data (total sample size n=101 683) revealed novel genome-wide significant association with MS susceptibility (p<5×10−8) for all seven variants. This included SNPs in or near LOC100506457 (rs1534422, p=4.03×10−12), CD28 (rs6435203, p=1.35×10−9), LPP (rs4686953, p=3.35×10−8), ETS1 (rs3809006, p=7.74×10−9), DLEU1 (rs806349, p=8.14×10−12), LPIN3 (rs6072343, p=7.16×10−12) and IFNGR2 (rs9808753, p=4.40×10−10). Cis expression quantitative locus effects were observed in silico for rs6435203 on CD28 and for rs9808753 on several immunologically relevant genes in the IFNGR2 locus. Conclusions: This study adds seven loci to the list of genuine MS genetic risk factors and further extends the list of established loci shared across autoimmune diseases

Genome-wide significant association with seven novel multiple sclerosis risk loci

Objective: A recent large-scale study in multiple sclerosis (MS) using the ImmunoChip platform reported on 11 loci that showed suggestive genetic association with MS. Additional data in sufficiently sized and independent data sets are needed to assess whether these loci represent genuine MS risk factors. Methods: The lead SNPs of all 11 loci were genotyped in 10 796 MS cases and 10 793 controls from Germany, Spain, France, the Netherlands, Austria and Russia, that were independent from the previously reported cohorts. Association analyses were performed using logistic regression based on an additive model. Summary effect size estimates were calculated using fixed-effect meta-analysis. Results: Seven of the 11 tested SNPs showed significant association with MS susceptibility in the 21 589 individuals analysed here. Meta-analysis across our and previously published MS case-control data (total sample size n=101 683) revealed novel genome-wide significant association with MS susceptibility (p<5×10−8) for all seven variants. This included SNPs in or near LOC100506457 (rs1534422, p=4.03×10−12), CD28 (rs6435203, p=1.35×10−9), LPP (rs4686953, p=3.35×10−8), ETS1 (rs3809006, p=7.74×10−9), DLEU1 (rs806349, p=8.14×10−12), LPIN3 (rs6072343, p=7.16×10−12) and IFNGR2 (rs9808753, p=4.40×10−10). Cis expression quantitative locus effects were observed in silico for rs6435203 on CD28 and for rs9808753 on several immunologically relevant genes in the IFNGR2 locus. Conclusions: This study adds seven loci to the list of genuine MS genetic risk factors and further extends the list of established loci shared across autoimmune diseases

Assessment of microRNA-related SNP effects in the 3′ untranslated region of the IL22RA2 risk locus in multiple sclerosis

Abstract Recent large-scale association studies have identified over 100 MS risk loci. One of these MS risk variants is single-nucleotide polymorphism (SNP) rs17066096, located 14 kb downstream of IL22RA2. IL22RA2 represents a compelling MS candidate gene due to the role of IL-22 in autoimmunity; however, rs17066096 does not map into any known functional element. We assessed whether rs17066096 or a nearby proxy SNP may exert pathogenic effects by affecting microRNA-to-mRNA binding and thus IL22RA2 expression using comprehensive in silico predictions, in vitro reporter assays, and genotyping experiments in 6,722 individuals. In silico screening identified two predicted microRNA binding sites in the 3′UTR of IL22RA2 (for hsa-miR-2278 and hsamiR-411-5p) encompassing a SNP (rs28366) in moderate linkage disequilibrium with rs17066096 (r 2 =0.4). The binding of both microRNAs to the IL22RA2 3′UTR was confirmed in vitro, but their binding affinities were not significantly affected by rs28366. Association analyses revealed significant Electronic supplementary material The online version of this articl

Cloning and characterization of receptor kinases from the EGF- and TGF ß- family from the fox tapeworm Echinococcus multilocularis

Rezeptorkinasen spielen eine wichtige Rolle in der Kommunikation von Zellen mit ihrer Umgebung und sind möglicherweise an hormonellen Kommunikations- Mechanismen zwischen parasitären Helminthen und ihren Säugetier- Wirten beteiligt. In dieser Arbeit wurden erstmals eine Rezeptor- Tyrosinkinase der EGF Familie, eine Serin- Threoninkinase der TGF-&#61538; Familie sowie ein intrazellulärer Signaltransduktionsfaktor der Smad- Familie aus dem Fuchsbandwurm Echinococcus multilocularis charakterisiert. Mittels degenerativer PCR und 3´/ 5´-RACE Methoden konnten drei E. multilocularis cDNAs identifiziert und vollständig charakterisiert werden, welche für (i) eine Tyrosinkinase (EmRTK1) der EGF Rezeptor- Familie (5160 bp cDNA, 1564 Aminosäuren); (ii) eine Serin – Threoninkinase (EmRSK1) der TGF-&#61538;&#61472;Rezeptor - Familie (1892 bp cDNA, 543 Aminosäuren); und (iii) einen intrazellulären Signaltransduktor der Smad Familie (1530 bp cDNA, 318Aminosäuren) kodieren. Anhand von Sequenzvergleichen der abgeleiteten Aminosäuresequenzen zeigten alle drei Faktoren für die jeweilige Proteinfamilie typische Domänenstrukturen und hohe Homologien zu bereits bekannten Faktoren aus Säugern. Der zugehörige chromosomale Locus wurde in allen drei Fällen vollständig charakterisiert. Mit Hilfe von RT-PCR Analysen konnte die Expression von emrtk-1, emrsk-1 und emsmadA in den Larvenstadien Metacestode und Protoskolex während der Infektion des Zwischenwirtes nachgewiesen werden. Anhand dieser Daten kann vermutet werden, dass die beschriebenen Rezeptoren und EmSmadA eine wichtige Rolle in der Entwicklung des Parasiten spielen und möglicherweise an Mechanismen der Wirt- Parasit Interaktion während der alveolären Echinokokkose beteiligt sind.Receptor kinases play an important role in the communication of cells with their environment and could be involved in hormonal communication- mechanisms between parasitic helminths and their mammalian hosts. This work describes, for the first time, the identification and characterization of a receptor- tyrosinkinase of the EGF- receptor family, a serin- threoninkinase of the TGF-&#61538;- receptor family, and an intracellular signal transduction factor of the Smad- Familie from the fox- tapeworm Echinococcus multilocularis. Using degenerative PCR and 3´/ 5´-RACE methods, E. multilocularis cDNAs coding for (i) a tyrosinkinase (EmRTK1) of the EGF family (5160 bp cDNA, 1564 amino acids); (ii) a serin – threoninkinase (EmRSK1) of the TGF-&#61538; family (1829 bp cDNA, 543 amino acids); and (iii) an intracellular signal-transductor of the Smad family (1530 bp cDNA, 318 amino acids) could be identified and completely characterized. Furthermore, the chromosomal loci for all three factors were completely characterized. Structural analyses revealed that all three Echinococcus factors displayed domain structures which are characteristic of the respective protein families. Furthermore, on the amino acid sequence level, all three factors shared considerable homologies with corresponding host factors. Using RT-PCR analysis, emrtk-1, emrsk-1 and emsmadA were shown to be expressed in the larval stages metacestode and protoscolex during the infection of the intermediate host. According to this data, the described receptors and EmSmadA are likely to play an important role in the parasitic development and could be involved in mechanisms of the host- parasite interaction during alveolar echinococcosis

Rolle von Immunzellen in hereditären Myelinopathien

Myelin mutations in the central and peripheral nervous system lead to severely disabling, currently untreatable diseases. In this study, we used transgenic PLP overexpressing mice (PLPtg) as a model for central inherited myelinopathies, such as leukodystrophies, and heterozygously P0 deficient (P0+/-) mice as models for peripheral hereditary polyneuropathies. Both models are characterized by low grade nervous tissue inflammation. Macrophages and CD8+ T- lymphocytes contribute to the myelin pathology as shown by crossbreeding experiments with immunodeficient mice. Having shown the relevance of CD8+ T- lymphocytes in PLPtg mice, we investigated the influence of one major cytotoxic molecule (granzyme B) on neural damage. By generation of granzyme B deficient PLPtg bone marrow chimeras, we could demonstrate a reduction of myelin pathology and oligodendrocyte death. Taken together, granzyme B is at least partly responsible for the cytotoxicity induced neural damage in PLPtg mice. To further explore the role of immune modulation, we focussed on the influence of the coinhibitory molecule PD-1, a CD28-related receptor expressed on activated T- and B-lymphocytes. By investigating myelin mutants of the CNS and PNS (PLPtg and P0+/-) with an additional PD-1 deficiency, induced by crossbreeding or bone marrow chimerization, we found a significant increase of CD8+ T- lymphocytes and massive increase of the myelin pathology in both the CNS and PNS model. In PLPtg mice, absence of PD-1 increased oligodendrocyte apoptosis, clonal expansions and a higher propensity of CNS but not peripheral CD8+ T- cells to secrete proinflammatory cytokines. In P0+/- mice, absence of PD-1 lead to moderate motor and sensory disturbances, confirming the important role of PD-1 in immune homeostasis. Taken together, we identified granzyme B as an important effector agent of cytotoxic T-lymphocytes in PLPtg mice and PD-1 as a crucial player in regulating the effector cells in our models of central and peripheral myelinopathy. Alterations of this regulatory pathway lead to overt neuroinflammation of high pathogenetic impact. These results might help to understand mechanisms responsible for high clinical variability of polygenic or even monogenic disorders of the nervous system.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

Microglia/macrophages are ultrastructurally altered by their proximity to spinal cord injury in adult female mice

Abstract Traumatic spinal cord injury can cause immediate physical damage to the spinal cord and result in severe neurological deficits. The primary, mechanical tissue damage triggers a variety of secondary damage mechanisms at the injury site which significantly contribute to a larger lesion size and increased functional damage. Inflammatory mechanisms which directly involve both microglia (MG) and monocyte-derived macrophages (MDM) play important roles in the post-injury processes, including inflammation and debris clearing. In the current study, we investigated changes in the structure and function of MG/MDM in the injured spinal cord of adult female mice, 7 days after a thoracic contusion SCI. With the use of chip mapping scanning electron microscopy, which allows to image large samples at the nanoscale, we performed an ultrastructural comparison of MG/MDM located near the lesion vs adjacent regions to provide novel insights into the mechanisms at play post-injury. We found that MG/MDM located near the lesion had more mitochondria overall, including mitochondria with and without morphological alterations, and had a higher proportion of altered mitochondria. MG/MDM near the lesion also showed an increased number of phagosomes, including phagosomes containing myelin and partiallydigested materials. MG/MDM near the injury interacted differently with the spinal cord parenchyma, as shown by their reduced number of direct contacts with synaptic elements, axon terminals and dendritic spines. In this study, we characterized the ultrastructural changes of MG/MDM in response to spinal cord tissue damage in mice, uncovering changes in phagocytic activity, mitochondrial ultrastructure, and inter-cellular interactions within the spinal cord parenchyma