Anne K. Mausberg

Murine Guanylate Binding Protein 2 (mGBP2) controls Toxoplasma gondii replication

IFN-γ orchestrates the host response against intracellular pathogens. Members of the guanylate binding proteins (GBP) comprise the most abundant IFN-γ–induced transcriptional response. mGBPs are GTPases that are specifically up-regulated by IFN-γ, other proinflammatory cytokines, toll-like receptor agonists, as well as in response to Listeria monocytogenes and Toxoplasma gondii infection. mGBP2 localizes at the parasitophorous vacuole (PV) of T. gondii; however, the molecular function of mGBP2 and its domains in T. gondii infection is not known. Here, we show that mGBP2 is highly expressed in several cell types, including T and B cells after stimulation. We provide evidence that the C-terminal domain is sufficient and essential for recruitment to the T. gondii PV. Functionally, mGBP2 reduces T. gondii proliferation because mGBP2-deficient cells display defects in the replication control of T. gondii. Ultimately, mGBP2-deficient mice reveal a marked immune susceptibility to T. gondii. Taken together, mGBP2 is an essential immune effector molecule mediating antiparasitic resistance.

Natalizumab exerts a suppressive effect on surrogates of B cell function in blood and CSF.

Background: Natalizumab for multiple sclerosis (MS) increases the risk of progressive multifocal leukoencephalopathy (PML). Objective: We aimed to assess the effect of natalizumab on cellular composition and functional B cell parameters including patients with natalizumab-associated PML (n=37). Methods: Cellular composition by flow cytometry, levels of immunoglobulin (Ig)G/IgM by immunonephelometry, and oligoclonal bands by isoelectric focusing were studied in blood and cerebrospinal fluid. Results: In MS patients treated with natalizumab without PML (n=59) the proportion of CD19+ B cells was higher in blood, but lower in cerebrospinal fluid compared with MS patients not treated with natalizumab (n=17). The CD4/CD8-ratio in cerebrospinal fluid was lower, and IgG and IgM levels as well as the IgG index dropped in longitudinal samples during natalizumab therapy. Oligoclonal bands persisted, but the total amount of the intrathecally produced IgG fraction, and the polyclonal intrathecal IgG reactivity to measles, rubella, and zoster declined. At the time of diagnosis of PML patients with natalizumab-associated PML had low total IgG levels in blood and cerebrospinal fluid. Conclusions: Natalizumab impacts B and T cell distribution and exerts an inhibitory effect on surrogates of B cell function in periphery and in cerebrospinal fluid, potentially contributing to the increased risk of developing PML.

Recovery of the T-cell repertoire in CIDP by IV immunoglobulins

Objective: To investigate changes in the T-cell repertoire in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) without and with treatment of IV immunoglobulins (IVIg). Methods: The T-cell receptor (TCR) repertoire of CD4+ and CD8+ T cells in the peripheral blood was analyzed using CDR3 spectratyping. Patients with CIDP were included without (n = 14) and with IVIg treatment (n = 11) cross-sectionally and longitudinally (n = 2). Results: While the TCR length distribution of patients with CIDP was only moderately altered for most of the Vβ elements of CD4+ T cells, the CD8+ population displayed extensive oligoclonal expansions in all analyzed 24 Vβ elements. A public expansion of a distinct TCR length in one Vβ element within a majority of affected patients was not detectable. Treatment with IVIg reduced the oligoclonal expansions within both the CD4+ and CD8+ population. Conclusions: Our data demonstrate that cytotoxic CD8+ T cells exhibit a much broader activation than CD4+ T cells, indicating a potentially crucial role of CD8+ T cells in the immunopathogenesis of CIDP. The profound oligoclonal response in T-cell activation suggests that multiple peptides may induce and propagate this autoimmune-driven disease. The observed reduction of highly activated T cells may contribute to the therapeutic effects of IVIg.

Natalizumab affects the T-cell receptor repertoire in patients with multiple sclerosis

Objective: To assess changes in the T-cell receptor (TCR) repertoire in peripheral venous blood and CSF of patients with multiple sclerosis (MS) treated with natalizumab and the potential implication for developing progressive multifocal leukoencephalopathy (PML) and PML–immune reconstitution inflammatory syndrome (IRIS). Methods: The TCR repertoire in blood and CSF was assessed by complementarity determining region 3 spectratyping in 59 patients with MS treated with natalizumab for at least 18 months, 5 cases of natalizumab-associated PML, 17 age- and sex-matched patients with MS not treated with natalizumab, and 12 healthy controls. Results: Patients with MS presented with peripheral TCR repertoire expansions in blood, which appeared less prominent during therapy with natalizumab. TCR repertoire restrictions observed in CSF were most pronounced in patients with MS treated with natalizumab. In patients who developed PML with longitudinal samples available, new identical TCR receptor length expansions in blood and CSF were observed following plasma exchange, and preceded the development of IRIS. Conclusions: Profound TCR repertoire restrictions in CSF of patients treated with natalizumab reflect an altered immune surveillance of the CNS, which may contribute to an increased risk of developing PML. Natalizumab seems to prompt an impaired or delayed peripheral expansion of antigen-specific T cells, whereas increased reconstitution of peripheral T-cell expansion following plasma exchange may trigger PML-IRIS. Our data suggest that treatment with natalizumab results in broader changes in the T-cell immune repertoire beyond lymphocyte migration.

Erythropoietin Ameliorates Rat Experimental Autoimmune Neuritis by Inducing Transforming Growth Factor-Beta in Macrophages

Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. In addition, in various preclinical models EPO exhibited protective activity against tissue injury. There is an urgent need for potent treatments of autoimmune driven disorders of the peripheral nervous system (PNS), such as the Guillain-Barré syndrome (GBS), a disabling autoimmune disease associated with relevant morbidity and mortality. To test the therapeutic potential of EPO in experimental autoimmune neuritis (EAN) - an animal model of human GBS – immunological and clinical effects were investigated in a preventive and a therapeutic paradigm. Treatment with EPO reduced clinical disease severity and if given therapeutically also shortened the recovery phase of EAN. Clinical findings were mirrored by decreased inflammation within the peripheral nerve, and myelin was well maintained in treated animals. In contrast, EPO increased the number of macrophages especially in later stages of the experimental disease phase. Furthermore, the anti-inflammatory cytokine transforming growth factor (TGF)-beta was upregulated in the treated cohorts. In vitro experiments revealed less proliferation of T cells in the presence of EPO and TGF-beta was moderately induced, while the secretion of other cytokines was almost not altered by EPO. Our data suggest that EPO revealed its beneficial properties by the induction of beneficial macrophages and the modulation of the immune system towards anti-inflammatory responses in the PNS. Further studies are warranted to elaborate the clinical usefulness of EPO for treating immune-mediated neuropathies in affected patients.

Levetiracetam exhibits protective properties on rat Schwann cells in vitro.

Oxidative stress and inflammation represent pathways causing substantial damage to the peripheral nervous system. Levetiracetam (LEV) is a commonly used antiepileptic drug targeting high‐voltage activated N‐type calcium channels. Recent evidence suggests that LEV may also act as a histone deacetylase inhibitor, suggesting that this drug exhibits both anti‐inflammatory and anti‐oxidative effects, and as such may represent an interesting candidate for treating inflammatory diseases affecting the peripheral nerve. Therefore, we analysed the influence of LEV ex vivo on purified Schwann cells from neonatal P3 rats as well as on dorsal root ganglia prepared from E15 rat embryos. LEV diminished a lipopolysaccharide (LPS)‐induced increase of the pro‐inflammatory signature molecules tumour necrosis factor alpha, matrix metalloproteinase 9 (MMP‐9), and caspase 6. Furthermore, LEV decreased LPS‐induced cell death and protected cells against oxidative stress in a glutamate‐based oxidative stress model. MMP‐2 activity, usually elevated during myelination and repair, was also found to be up‐regulated following LEV, while LEV exhibited no negative effects on myelination. Intracellular sodium or calcium concentrations were unaltered by LEV. Thus, LEV may be a promising, well‐tolerated drug that – besides its antiepileptic potential – mediates anti‐inflammatory, anti‐oxidative, and anti‐apoptotic properties that may potentially be useful in treating diseases of the peripheral nerve.

Intracerebral granulocyte-macrophage colony-stimulating factor induces functionally competent dendritic cells in the mouse brain.

Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is a hematopoietic growth factor and a proinflammatory cytokine. While GM‐CSF is lacking in normal brain tissue, it is expressed under pathological conditions and correlates with the presence of dendritic cells (DC). However, the role of GM‐CSF for the onset of immune responses in the brain is still unclear. To analyze the role of GM‐CSF for the induction and functional activity of immune cells in the brain, we performed chronic intracerebroventricular administration of GM‐CSF to the brains of adult mice. After GM‐CSF administration, intracerebral leukocytes (ICL) were characterized by means of flow cytometry, immunohistochemistry, and an ex vivo functional assay. GM‐CSF treatment significantly increased the number of leukocytes expressing high levels of CD45, indicative of peripheral, blood‐derived cells. The infiltrating cells were preferentially DC of the myeloid lineage (CD45high CD11c+ CD11b+) with an activated phenotype characterized by upregulated expression of MHCII and the costimulatory ligand CD80. Furthermore, DC from GM‐CSF treated mice were fully competent to activate naive allogeneic T cells in a mixed leukocyte reaction. In contrast, intracerebroventricular IFN‐γ administration stimulated MHCII expression on cells resembling resident microglia, but did not induce comparable presence of DC. Taken together, intracerebroventricular GM‐CSF treatment results in high numbers of DC in the brain. Moreover, these GM‐CSF‐induced DC display an activated phenotype and exhibit the capacity to act as fully competent DC even without a further inflammatory stimulus.

FoxP3+ Regulatory T Cells Determine Disease Severity in Rodent Models of Inflammatory Neuropathies

Inflammatory neuropathies represent disabling human autoimmune disorders with considerable disease variability. Animal models provide insights into defined aspects of their disease pathogenesis. Forkhead box P3 (FoxP3)+ regulatory T lymphocytes (Treg) are anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. Dysfunction or a reduced frequency of Tregs have been associated with different human autoimmune disorders. We here analyzed the functional relevance of Tregs in determining disease manifestation and severity in murine models of autoimmune neuropathies. We took advantage of the DEREG mouse system allowing depletion of Treg with high specificity as well as anti-CD25 directed antibodies to deplete Tregs in mice in actively induced experimental autoimmune neuritis (EAN). Furthermore antibody-depletion was performed in an adoptive transfer model of chronic neuritis. Early Treg depletion increased clinical EAN severity both in active and adoptive transfer chronic neuritis. This was accompanied by increased proliferation of myelin specific T cells and histological signs of peripheral nerve inflammation. Late stage Treg depletion after initial disease manifestation however did not exacerbate inflammatory neuropathy symptoms further. We conclude that Tregs determine disease severity in experimental autoimmune neuropathies during the initial priming phase, but have no major disease modifying function after disease manifestation. Potential future therapeutic approaches targeting Tregs should thus be performed early in inflammatory neuropathies.

Mouse Schwann cells activate MHC class I and II restricted T-cell responses, but require external peptide processing for MHC class II presentation

Schwann cells are the myelinating glia cells of the peripheral nervous system (PNS). In inflammatory neuropathies like the Guillain-Barré syndrome (GBS) Schwann cells become target of an autoimmune response, but may also modulate local inflammation. Here, we tested the functional relevance of Schwann cell derived MHC expression in an in vitro coculture system. Mouse Schwann cells activated proliferation of ovalbumin specific CD8+ T cells when ovalbumin protein or MHC class I restricted ovalbumin peptide (Ova(257-264) SIINFEKL) was added and after transfection with an ovalbumin coding vector. Schwann cells activated proliferation of ovalbumin specific CD4+ T cells in the presence of MHC class II restricted ovalbumin peptide (Ova(323-339) ISQAVHAAHAEINEAGR). CD4+ T-cell proliferation was not activated by ovalbumin protein or transfection with an ovalbumin coding vector. This indicates that Schwann cells express functionally active MHC class I and II molecules. In this study, however, Schwann cells lacked the ability to process exogenous antigen or cross-present endogenous antigen into the MHC class II presentation pathway. Thus, antigen presentation may be a pathological function of Schwann cells exacerbating nerve damage in inflammatory neuropathies.

Thymic Epithelium Determines a Spontaneous Chronic Neuritis in Icam1 tm1Jcgr NOD Mice

The NOD mouse strain spontaneously develops autoimmune diabetes. A deficiency in costimulatory molecules, such as B7-2, on the NOD genetic background prevents diabetes but instead triggers an inflammatory peripheral neuropathy. This constitutes a shift in the target of autoimmunity, but the underlying mechanism remains unknown. In this study, we demonstrate that NOD mice deficient for isoforms of ICAM-1, which comediate costimulatory functions, spontaneously develop a chronic autoimmune peripheral neuritis instead of diabetes. The disease is transferred by CD4+ T cells, which infiltrate peripheral nerves together with macrophages and B cells and are autoreactive against peripheral myelin protein zero. These Icam1tm1JcgrNOD mice exhibit unaltered numbers of regulatory T cells, but increased IL-17–producing T cells, which determine the severity, but not the target specificity, of autoimmunity. Ab-mediated ICAM-1 blockade triggers neuritis only in young NOD mice. Thymic epithelium from Icam1tm1JcgrNOD mice features an altered expression of costimulatory molecules and induces neuritis and myelin autoreactivity after transplantation into nude mice in vivo. Icam1tm1JcgrNOD mice exhibit a specifically altered TCR repertoire. Our findings introduce a novel animal model of chronic inflammatory neuropathies and indicate that altered expression of ICAM-1 on thymic epithelium shifts autoimmunity specifically toward peripheral nerves. This improves our understanding of autoimmunity in the peripheral nervous system with potential relevance for human diseases.