Pro-inflammatory T helper 17 directly harms oligodendrocytes in neuroinflammation.

T helper (Th)17 cells are considered to contribute to inflammatory mechanisms in diseases such as multiple sclerosis (MS). However, the discussion persists regarding their true role in patients. Here, we visualized central nervous system (CNS) inflammatory processes in models of MS live in vivo and in MS brains and discovered that CNS-infiltrating Th17 cells form prolonged stable contact with oligodendrocytes. Strikingly, compared to Th2 cells, direct contact with Th17 worsened experimental demyelination, caused damage to human oligodendrocyte processes, and increased cell death. Importantly, we found that in comparison to Th2 cells, both human and murine Th17 cells express higher levels of the integrin CD29, which is linked to glutamate release pathways. Of note, contact of human Th17 cells with oligodendrocytes triggered release of glutamate, which induced cell stress and changes in biosynthesis of cholesterol and lipids, as revealed by single-cell RNA-sequencing analysis. Finally, exposure to glutamate decreased myelination, whereas blockade of CD29 preserved oligodendrocyte processes from Th17-mediated injury. Our data provide evidence for the direct and deleterious attack of Th17 cells on the myelin compartment and show the potential for therapeutic opportunities in MS.

Gatekeeper role of brain antigen-presenting CD11c+ cells in neuroinflammation.

Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen-presenting cells, dendritic cells. Applying intravital two-photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen-presenting CD11c(+) cells, which preferentially interact with Th17 cells. IL-17 expression correlates with expression of GM-CSF by T cells and with accumulation of CNS CD11c(+) cells. These CD11c(+) cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c(+) cells in the attraction of pathogenic T cells into and their survival within the CNS. Depletion of CD11c(+) cells markedly reduced disease severity due to impaired enrichment of pathogenic T cells within the CNS.

Cross-recognition of a myelin peptide by CD8+ T cells in the CNS is not sufficient to promote neuronal damage.

Multiple sclerosis (MS) is an inflammatory disease of the CNS thought to be driven by CNS-specific T lymphocytes. Although CD8(+) T cells are frequently found in multiple sclerosis lesions, their distinct role remains controversial because direct signs of cytotoxicity have not been confirmed in vivo. In the present work, we determined that murine ovalbumin-transgenic (OT-1) CD8(+) T cells recognize the myelin peptide myelin oligodendrocyte glycoprotein 40-54 (MOG40-54) both in vitro and in vivo. The aim of this study was to investigate whether such cross-recognizing CD8(+) T cells are capable of inducing CNS damage in vivo. Using intravital two-photon microscopy in the mouse model of multiple sclerosis, we detected antigen recognition motility of the OT-1 CD8(+) T cells within the CNS leading to a selective enrichment in inflammatory lesions. However, this cross-reactivity of OT-1 CD8(+) T cells with MOG peptide in the CNS did not result in clinically or subclinically significant damage, which is different from myelin-specific CD4(+) Th17-mediated autoimmune pathology. Therefore, intravital imaging demonstrates that local myelin recognition by autoreactive CD8(+) T cells in inflammatory CNS lesions alone is not sufficient to induce disability or increase axonal injury.

In vivo and in vitro effects of multiple sclerosis immunomodulatory therapeutics on glutamatergic excitotoxicity.

In multiple sclerosis (MS), a candidate downstream mechanism for neuronal injury is glutamate (Glu)-induced excitotoxicity, leading to toxic increases in intraneuronal Ca(2+) . Here, we used in vivo two-photon imaging in the brain of TN-XXL transgenic Ca(2+) reporter mice to test whether promising oral MS therapeutics, namely fingolimod, dimethyl fumarate, and their respective metabolites fingolimod-phosphate and monomethyl fumarate, can protect neurons against acute glutamatergic excitotoxic damage. We also assessed whether these drugs can protect against excitotoxicity in vitro using primary cortical neurons, and whether they can directly inhibit Glu release from pathogenic T-helper 17 lymphocytes. In vivo, direct and acute (1 h) administration of 100 mM Glu to the brainstem resulted in a rapid and significant up-regulation in neuronal Ca(2+) signaling as well as morphological excitotoxic changes that were attenuated by the NMDA-receptor antagonist MK801. Direct CNS administration of MS drugs prior to Glu significantly delayed or reduced, but did not prevent the neuronal Ca(2+) increase or morphological changes. In vitro, prolonged (24 h) treatment of primary neurons with the fumarates significantly protected against neurotoxicity induced by Glu as well as NMDA, similar to MK801. Furthermore, monomethyl fumerate significantly reduced Glu release from pathogenic T-helper 17 lymphocytes. Overall, these data suggest that MS drugs may mediate neuroprotection via excitotoxicity modulating effects. Evidence suggests MS pathogenesis may involve neuronal excitotoxicity, induced by local release of glutamate. However, current MS drugs, including dimethyl fumerate (DMF) and fingolimod (FTY720) are largely anti-inflammatory and not yet fully tested for their neuroprotective potential. Here, we show that the drugs, in particular DMF metabolite monomethyl fumerate (MMF), protect neurons by excitotoxicity modulating effects. Th17, T-helper 17.

Perivascular microglia promote blood vessel disintegration in the ischemic penumbra.

The contribution of microglia to ischemic cortical stroke is of particular therapeutic interest because of the impact on the survival of brain tissue in the ischemic penumbra, a region that is potentially salvable upon a brain infarct. Whether or not tissue in the penumbra survives critically depends on blood flow and vessel perfusion. To study the role of microglia in cortical stroke and blood vessel stability, CX3CR1(+/GFP) mice were subjected to transient middle cerebral artery occlusion and then microglia were investigated using time-lapse two-photon microscopy in vivo. Soon after reperfusion, microglia became activated in the stroke penumbra and started to expand cellular protrusions towards adjacent blood vessels. All microglia in the penumbra were found associated with blood vessels within 24 h post reperfusion and partially fully engulfed them. In the same time frame blood vessels became permissive for blood serum components. Migration assays in vitro showed that blood serum proteins leaking into the tissue provided molecular cues leading to the recruitment of microglia to blood vessels and to their activation. Subsequently, these perivascular microglia started to eat up endothelial cells by phagocytosis, which caused an activation of the local endothelium and contributed to the disintegration of blood vessels with an eventual break down of the blood brain barrier. Loss-of-microglia-function studies using CX3CR1(GFP/GFP) mice displayed a decrease in stroke size and a reduction in the extravasation of contrast agent into the brain penumbra as measured by MRI. Potentially, medication directed at inhibiting microglia activation within the first day after stroke could stabilize blood vessels in the penumbra, increase blood flow, and serve as a valuable treatment for patients suffering from ischemic stroke.

In vivo imaging of lymphocytes in the CNS reveals different behaviour of naïve T cells in health and autoimmunity.

BACKGROUND: Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated. METHODS: We applied ex vivo and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures. RESULTS: By applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks. CONCLUSIONS: The activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS.

Multiphoton microscopy in the diagnostic assessment of pediatric solid tissue in comparison to conventional histopathology: results of the first international online interobserver trial.

Purpose: Clear resection margins are paramount for good outcome in children undergoing solid tumor resections. Multiphoton microscopy (MPM) can provide high-resolution, real-time, intraoperative microscopic images of tumor tissue. Objective: This prospective international multicenter study evaluates the diagnostic accuracy, feasibility, and interobserver congruence of MPM in diagnosing solid pediatric tissue and tumors for the first time. Material and methods: Representative fresh sections from six different neonatal solid tissues (liver, lung, kidney, adrenal gland, heart muscle, testicle) and two types of typical pediatric solid tumors (neuroblastoma, rhabdomyosarcoma) with adjacent nonneoplastic tissue were imaged with MPM and then presented online with corresponding H&E stained slides of the exact same tissue region. Both image sets of each tissue type were interpreted by 38 randomly selected international attending pediatric pathologists via an online evaluation software. Results: The quality of MPM was sufficient to make the diagnosis of all normal tissue types except cardiac muscle in >94% of assessors with high interobserver congruence and 95% sensitivity. Heart muscle was interpreted as skeletal muscle in 55% of cases. Based on MPM imaging, participating pathologists diagnosed the presented pediatric neoplasms with 100% specificity, although the sensitivity reached only about 50%. Conclusion: Even without prior training, pathologists are able to diagnose normal pediatric tissues with valuable accuracy using MPM. While current MPM imaging protocols are not yet sensitive enough to reliably rule out neuroblastoma or rhabdomyosarcoma, they seem to be specific and therefore useful to confirm a diagnosis intraoperatively. We are confident that improved algorithms, specific training, and more experience with the method will make MPM a valuable future alternative to frozen section analysis. Registration: The trial was registered at www.researchregistry.com, registration number 2967.

Fast direct neuronal signaling via the IL-4 receptor as therapeutic target in neuroinflammation.

Ongoing axonal degeneration is thought to underlie disability in chronic neuroinflammation, such as multiple sclerosis (MS), especially during its progressive phase. Upon inflammatory attack, axons undergo pathological swelling, which can be reversible. Because we had evidence for beneficial effects of T helper 2 lymphocytes in experimental neurotrauma and discovered interleukin-4 receptor (IL-4R) expressed on axons in MS lesions, we aimed at unraveling the effects of IL-4 on neuroinflammatory axon injury. We demonstrate that intrathecal IL-4 treatment during the chronic phase of several experimental autoimmune encephalomyelitis models reversed disease progression without affecting inflammation. Amelioration of disability was abrogated upon neuronal deletion of IL-4R. We discovered direct neuronal signaling via the IRS1-PI3K-PKC pathway underlying cytoskeletal remodeling and axonal repair. Nasal IL-4 application, suitable for clinical translation, was equally effective in improving clinical outcome. Targeting neuronal IL-4 signaling may offer new therapeutic strategies to halt disability progression in MS and possibly also neurodegenerative conditions.

Phenotype of Antigen Unexperienced TH Cells in the Inflamed Central Nervous System in Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis is a chronic, disseminated inflammation of the central nervous system which is thought to be driven by autoimmune T cells. Genetic association studies in multiple sclerosis and a large number of studies in the animal model of the disease support a role for effector/memory T helper cells. However, the mechanisms underlying relapses, remission and chronic progression in multiple sclerosis or the animal model experimental autoimmune encephalomyelitis, are not clear. In particular, there is only scarce information on the role of central nervous system-invading naive T helper cells in these processes. By applying two-photon laser scanning microscopy we could show in vivo that antigen unexperienced T helper cells migrated into the deep parenchyma of the inflamed central nervous system in experimental autoimmune encephalomyelitis, independent of their antigen specificity. Using flow cytometric analyses of central nervous system-derived lymphocytes we found that only antigen-specific, formerly naive T helper cells became activated during inflammation of the central nervous system encountering their corresponding antigen.

Multiphoton microscopy: A novel diagnostic method for solid tumors in a prospective pediatric oncologic cohort, an experimental study.

BACKGROUND: The prognosis of solid pediatric tumors strongly correlates with accurate staging and complete local control. Currently, surgeons rely on macroscopic cues and intraoperative cryosection to determine resection borders. Multiphoton Microscopy (MPM) is a real time technique that allows imaging of tissue without time-consuming tissue processing. PURPOSE: This pilot study evaluates the diagnostic potential of MPM in pediatric solid tumors compared to routine histopathology. METHODS: Slides of pediatric tumor samples (nephroblastoma and neuroblastoma [n = 2]; ganglioneuroma, pleuropulmonary blastoma, hepatocellular carcinoma [n = 1]) were prepared to allow direct comparison of MPM with conventional light microscopy. Additionally, we applied MPM to native tumor tissue blocks to evaluate direct visualization of malignant cells through the tumor capsule. Images were interpreted by an attending surgical pathologist. Detectability of tumor-specific features was compared between MPM and conventional histology. RESULTS: A total of 7 tumors from 7 recruited patients were analyzed. All MPM images were accurate in diagnosing typical criteria of each particular neoplasm. In addition, MPM clearly visualized tumors through the capsule without sectioning or labeling procedures. The quality of MPM was sufficient to make the diagnosis and visualize typical entity-specific architectural changes. CONCLUSION: MPM is comparable to conventional histopathology in the diagnosis of pediatric solid tumors without the need for fixation or staining. It therefore has tremendous potential for future real-time intraoperative diagnostics and as an alternative to conventional frozen section histopathology. LEVEL OF EVIDENCE: III.

FRET based ratiometric Ca(2+) imaging to investigate immune-mediated neuronal and axonal damage processes in experimental autoimmune encephalomyelitis.

BACKGROUND: Irreversible axonal and neuronal damage are the correlate of disability in patients suffering from multiple sclerosis (MS). A sustained increase of cytoplasmic free [Ca(2+)] is a common upstream event of many neuronal and axonal damage processes and could represent an early and potentially reversible step. NEW METHOD: We propose a method to specifically analyze the neurodegenerative aspects of experimental autoimmune encephalomyelitis by Förster Resonance Energy Transfer (FRET) imaging of neuronal and axonal Ca(2+) dynamics by two-photon laser scanning microscopy (TPLSM). RESULTS: Using the genetically encoded Ca(2+) sensor TN-XXL expressed in neurons and their corresponding axons, we confirm the increase of cytoplasmic free [Ca(2+)] in axons and neurons of autoimmune inflammatory lesions compared to those in non-inflamed brains. We show that these relative [Ca(2+)] increases were associated with immune-neuronal interactions. COMPARISON WITH EXISTING METHODS: In contrast to Ca(2+)-sensitive dyes the use of a genetically encoded Ca(2+) sensor allows reliable intraaxonal free [Ca(2+)] measurements in living anesthetized mice in health and disease. This method detects early axonal damage processes in contrast to e.g. cell/axon morphology analysis, that rather detects late signs of neurodegeneration. CONCLUSIONS: Thus, we describe a method to analyze and monitor early neuronal damage processes in the brain in vivo.

Existence of long-lasting experience-dependent plasticity in endocrine cell networks.

Experience-dependent plasticity of cell and tissue function is critical for survival by allowing organisms to dynamically adjust physiological processes in response to changing or harsh environmental conditions. Despite the conferred evolutionary advantage, it remains unknown whether emergent experience-dependent properties are present in cell populations organized as networks within endocrine tissues involved in regulating body-wide homeostasis. Here we show, using lactation to repeatedly activate a specific endocrine cell network in situ in the mammalian pituitary, that templates of prior demand are permanently stored through stimulus-evoked alterations to the extent and strength of cell-cell connectivity. Strikingly, following repeat stimulation, evolved population behaviour leads to improved tissue output. As such, long-lasting experience-dependent plasticity is an important feature of endocrine cell networks and underlies functional adaptation of hormone release.

Influence of estrogens on GH-cell network dynamics in females: a live in situ imaging approach.

The secretion of endocrine hormones from pituitary cells finely regulates a multitude of homeostatic processes. To dynamically adapt to changing physiological status and environmental stimuli, the pituitary gland must undergo marked structural and functional plasticity. Endocrine cell plasticity is thought to primarily rely on variations in cell proliferation and size. However, cell motility, a process commonly observed in a variety of tissues during development, may represent an additional mechanism to promote plasticity within the adult pituitary gland. To investigate this, we used multiphoton time-lapse imaging methods, GH-enhanced green fluorescent protein transgenic mice and sexual dimorphism of the GH axis as a model of divergent tissue demand. Using these methods to acutely (12 h) track cell dynamics, we report that ovariectomy induces a dramatic and dynamic increase in cell motility, which is associated with gross GH-cell network remodeling. These changes can be prevented by estradiol supplementation and are associated with enhanced network connectivity as evidenced by increased coordinated GH-cell activity during multicellular calcium recordings. Furthermore, cell motility appears to be sex-specific, because reciprocal alterations are not detected in males after castration. Therefore, GH-cell motility appears to play an important role in the structural and functional pituitary plasticity, which is evoked in response to changing estradiol concentrations in the female.