Bettina Schreiner

IL-9 as a mediator of Th17-driven inflammatory disease

We report that like other T cells cultured in the presence of transforming growth factor (TGF) β, Th17 cells also produce interleukin (IL) 9. Th17 cells generated in vitro with IL-6 and TGF-β as well as purified ex vivo Th17 cells both produced IL-9. To determine if IL-9 has functional consequences in Th17-mediated inflammatory disease, we evaluated the role of IL-9 in the development and progression of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. The data show that IL-9 neutralization and IL-9 receptor deficiency attenuates disease, and this correlates with decreases in Th17 cells and IL-6–producing macrophages in the central nervous system, as well as mast cell numbers in the regional lymph nodes. Collectively, these data implicate IL-9 as a Th17-derived cytokine that can contribute to inflammatory disease.

The Cytokine GM-CSF Drives the Inflammatory Signature of CCR2+ Monocytes and Licenses Autoimmunity

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine produced by auto-reactive T helper (Th) cells that initiate tissue inflammation. Multiple cell types can sense GM-CSF, but the identity of the pathogenic GM-CSF-responsive cells is unclear. By using conditional gene targeting, we systematically deleted the GM-CSF receptor (Csf2rb) in specific subpopulations throughout the myeloid lineages. Experimental autoimmune encephalomyelitis (EAE) progressed normally when either classical dendritic cells (cDCs) or neutrophils lacked GM-CSF responsiveness. The development of tissue-invading monocyte-derived dendritic cells (moDCs) was also unperturbed upon Csf2rb deletion. Instead, deletion of Csf2rb in CCR2(+)Ly6C(hi) monocytes phenocopied the EAE resistance seen in complete Csf2rb-deficient mice. High-dimensional analysis of tissue-infiltrating moDCs revealed that GM-CSF initiates a combination of inflammatory mechanisms. These results indicate that GM-CSF signaling controls a pathogenic expression signature in CCR2(+)Ly6C(hi) monocytes and their progeny, which was essential for tissue damage.

Modeling multiple sclerosis in laboratory animals

Inflammatory demyelinating disease of the central nervous system is one of the most frequent causes of neurological disability in young adults. While in situ analysis and in vitro models do shed some light onto the processes of tissue damage and cellular interactions, the development of neuroinflammation and demyelination is a far too complex process to be adequately modeled by simple test tube systems. Thus, animal models using primarily genetically modified mice have been proven to be of paramount importance. In this chapter, we discuss recent advances in modeling brain diseases focusing on murine models and report on new tools to study the pathogenesis of complex diseases such as multiple sclerosis.

High-Dimensional Single-Cell Mapping of Central Nervous System Immune Cells Reveals Distinct Myeloid Subsets in Health, Aging, and Disease

Summary Individual reports suggest that the central nervous system (CNS) contains multiple immune cell types with diverse roles in tissue homeostasis, immune defense, and neurological diseases. It has been challenging to map leukocytes across the entire brain, and in particular in pathology, where phenotypic changes and influx of blood‐derived cells prevent a clear distinction between reactive leukocyte populations. Here, we applied high‐dimensional single‐cell mass and fluorescence cytometry, in parallel with genetic fate mapping systems, to identify, locate, and characterize multiple distinct immune populations within the mammalian CNS. Using this approach, we revealed that microglia, several subsets of border‐associated macrophages and dendritic cells coexist in the CNS at steady state and exhibit disease‐specific transformations in the immune microenvironment during aging and in models of Alzheimer’s disease and multiple sclerosis. Together, these data and the described framework provide a resource for the study of disease mechanisms, potential biomarkers, and therapeutic targets in CNS disease. Graphical Abstract Figure. No Caption available. HighlightsHigh‐dimensional cytometry reveals diverse immune cells in the steady‐state CNSCD38 and MHCII distinguish CNS border‐associated macrophage (BAM) subsetsA subset of microglia responds to aging and neurodegenerationAll microglia are homogenously affected in neuroinflammatory disease &NA; It has been challenging to map leukocytes in the brain, particularly during pathology. Mrdjen et al. combine high‐dimensional single‐cell cytometry with fate mapping to capture the immune landscape of the brain. They identify different subsets of myeloid cells and the phenotypic changes in CNS immune cells during aging and in models of Alzheimer’s disease and multiple sclerosis.

IL-12 protects from psoriasiform skin inflammation.

Neutralization of the common p40-subunit of IL-12/23 in psoriasis patients has led to a breakthrough in the management of moderate to severe disease. Aside from neutralizing IL-23, which is thought to be responsible for the curative effect, anti-p40 therapy also interferes with IL-12 signalling and type 1 immunity. Here we dissect the individual contribution of these two cytokines to the formation of psoriatic lesions and understand the effect of therapeutic co-targeting of IL-12 and IL-23 in psoriasis. Using a preclinical model for psoriatic plaque formation we show that IL-12, in contrast to IL-23, has a regulatory function by restraining the invasion of an IL-17-committed γδT (γδT17) cell subset. We discover that IL-12 receptor signalling in keratinocytes initiates a protective transcriptional programme that limits skin inflammation, suggesting that collateral targeting of IL-12 by anti-p40 monoclonal antibodies is counterproductive in the therapy of psoriasis.

Astrocyte Depletion Impairs Redox Homeostasis and Triggers Neuronal Loss in the Adult CNS.

Although the importance of reactive astrocytes during CNS pathology is well established, the function of astroglia in adult CNS homeostasis is less well understood. With the use of conditional, astrocyte-restricted protein synthesis termination, we found that selective paralysis of GFAP(+) astrocytes in vivo led to rapid neuronal cell loss and severe motor deficits. This occurred while structural astroglial support still persisted and in the absence of any major microvascular damage. Whereas loss of astrocyte function did lead to microglial activation, this had no impact on the neuronal loss and clinical decline. Neuronal injury was caused by oxidative stress resulting from the reduced redox scavenging capability of dysfunctional astrocytes and could be prevented by the in vivo treatment with scavengers of reactive oxygen and nitrogen species (ROS/RNS). Our results suggest that the subpopulation of GFAP(+) astrocytes maintain neuronal health by controlling redox homeostasis in the adult CNS.

Dysregulation of the Cytokine GM-CSF Induces Spontaneous Phagocyte Invasion and Immunopathology in the Central Nervous System

SUMMARY Chronic inflammatory diseases are influenced by dysregulation of cytokines. Among them, granulocyte macrophage colony stimulating factor (GM‐CSF) is crucial for the pathogenic function of T cells in preclinical models of autoimmunity. To study the impact of dysregulated GM‐CSF expression in vivo, we generated a transgenic mouse line allowing the induction of GM‐CSF expression in mature, peripheral helper T (Th) cells. Antigen‐independent GM‐CSF release led to the invasion of inflammatory myeloid cells into the central nervous system (CNS), which was accompanied by the spontaneous development of severe neurological deficits. CNS‐invading phagocytes produced reactive oxygen species and exhibited a distinct genetic signature compared to myeloid cells invading other organs. We propose that the CNS is particularly vulnerable to the attack of monocyte‐derived phagocytes and that the effector functions of GM‐CSF‐expanded myeloid cells are in turn guided by the tissue microenvironment. Graphical Abstract Figure. No Caption available. HighlightsDysregulated GM‐CSF leads to the expansion of myeloid cellsGM‐CSF‐expanded phagocytes cause CNS inflammation and neurological deficitsCNS‐invading phagocytes produce reactive oxygen speciesTissue‐invading phagocytes exhibit a unique genetic signature in the CNS &NA; GM‐CSF is a pro‐inflammatory cytokine that mediates tissue‐targeted autoimmune diseases. Spath and colleagues show that excess production of GM‐CSF was sufficient to induce spontaneous brain inflammation and neurological disease. GM‐CSF‐expanded, brain‐infiltrating phagocytes produced reactive oxygen species and their genetic signature differed significantly from myeloid cells invading other organs.

Mature oligodendrocytes actively increase in vivo cytoskeletal plasticity following CNS damage

BackgroundOligodendrocytes are myelinating cells of the central nervous system which support functionally, structurally, and metabolically neurons. Mature oligodendrocytes are generally believed to be mere targets of destruction in the context of neuroinflammation and tissue damage, but their real degree of in vivo plasticity has become a matter of debate. We thus investigated the in vivo dynamic, actin-related response of these cells under different kinds of demyelinating stress.MethodsWe used a novel mouse model (oLucR) expressing luciferase in myelin oligodendrocyte glycoprotein-positive oligodendrocytes under the control of a β-actin promoter. Activity of this promoter served as surrogate for dynamics of the cytoskeleton gene transcription through recording of in vivo bioluminescence following diphtheria toxin-induced oligodendrocyte death and autoimmune demyelination. Cytoskeletal gene expression was quantified from mature oligodendrocytes directly isolated from transgenic animals through cell sorting.ResultsExperimental demyelinating setups augmented oligodendrocyte-specific in vivo bioluminescence. These changes in luciferase signal were confirmed by further ex vivo analysis of the central nervous system tissue from oLucR mice. Increase in bioluminescence upon autoimmune inflammation was parallel to an oligodendrocyte-specific increased transcription of β-tubulin.ConclusionsMature oligodendrocytes acutely increase their cytoskeletal plasticity in vivo during demyelination. They are therefore not passive players under demyelinating conditions but can rather react dynamically to external insults.

Perspectives on cytokine-directed therapies in multiple sclerosis.

Multiple sclerosis (MS) is the most common inflammatory demyelinating disorder of the central nervous system (CNS). Over the past 10 years there has been a heated debate as to whether MS pathogenesis commences in the CNS or whether it is actually primarily a disease of the immune system. The combined clinical data, therapy responses, pathology, animal models and genetic studies now provide overwhelming support for the concept that MS is a disease of the immune system and that the CNS is only the unfortunate target of a misguided immune attack. Immune cells communicate through the use of cytokines and these proteins can orchestrate the most complex behaviour in immune cells. We propose that MS is a disease where immune communication is derailed, which makes MS very amenable to immunotherapy and in particular makes cytokines an attractive target to repair this miscommunication disorder.

Loss of IGF1R from oligodendrocytes ameliorates neuroinflammation without affecting cell survival

Optic neuritis (ON) is an early syndrome present in patients with multiple sclerosis (MS). It is defined as an autoimmune demyelinating disorder that results in axonal loss and visual disturbances. In order to characterize axonal changes during ON, we used an animal model of MS, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in brown Norway rats. In this model, retinal ganglion cell (RGC) body loss is visible before inflammation of the optic nerve, suggesting that insult to the RGC body might be a primary event that triggers axonal stress, independent of later axonal loss resulting from inflammatory demyelination. This is supported by observations of disturbed axonal ultrastructure prior to demyelination by electron microscopy. In addition, changes in calcium homeostasis during the preclinical phase of the disease are seen which correlate with calpain-mediated cleavage of spectrin, a protein responsible for actin anchoring to the cell membrane and involved in actin remodeling. Therefore, this study aims to determine the timing of disturbances in axonal transport and cytoskeletal integrity, with focus particularly on the preclinical phase of ON. To achieve this, the following techniques have been employed: (1) immunohistochemistry to assess accumulation of various proteins associated with axonal transport (e.g. kinesin and synaptophysin), including the acute axonal injury marker, beta-APP; (2) intravitreal injections of cholera toxin B-FITC, a protein which is retroand anterogradely transported along axons, in order to monitor transport timing; and (3) assays to determine filamentous vs globular actin ratios in the optic nerve, to monitor actin destructuralization. Data obtained so far, indicate that despite evidence of changes in the actin cytoskeletal network and axonal ultrastructure during the preclinical disease stage, the microtubule assembly is not grossly affected, with no detectable perturbations in axonal transport. Instead, axonal transport failure is only seen during the clinical disease phase in the vicinity of demyelinating inflammatory lesions.