Multiple sclerosis (MS) is the most common chronic central nervous system inflammatory disease. Individual courses are highly variable, with complete remission in some patients and relentless ...progression in others. We generated induced pluripotent stem cells (iPSCs) to investigate possible mechanisms in benign MS (BMS), compared with progressive MS (PMS). We differentiated neurons and astrocytes that were then stressed with inflammatory cytokines typically associated with MS phenotypes. TNF-α/IL-17A treatment increased neurite damage in MS neurons from both clinical phenotypes. In contrast, TNF-α/IL-17A-reactive BMS astrocytes cultured with healthy control neurons exhibited less axonal damage compared with PMS astrocytes. Accordingly, single-cell transcriptomic BMS astrocyte analysis of cocultured neurons revealed upregulated neuronal resilience pathways; these astrocytes showed differential growth factor expression. Furthermore, supernatants from BMS astrocyte/neuronal cocultures rescued TNF-α/IL-17-induced neurite damage. This process was associated with a unique LIF and TGF-β1 growth factor expression, as induced by TNF-α/IL-17 and JAK-STAT activation. Our findings highlight a potential therapeutic role of modulation of astrocyte phenotypes, generating a neuroprotective milieu. Such effects could prevent permanent neuronal damage.
Neuronal damage in autoimmune neuroinflammation is the correlate for long-term disability in multiple sclerosis (MS) patients. Here, we investigated the role of immune cells in neuronal damage ...processes in animal models of MS by monitoring experimental autoimmune encephalomyelitis (EAE) by using two-photon microscopy of living anaesthetized mice. In the brainstem, we detected sustained interaction between immune and neuronal cells, particularly during disease peak. Direct interaction of myelin oligodendrocyte glycoprotein (MOG)-specific Th17 and neuronal cells in demyelinating lesions was associated with extensive axonal damage. By combining confocal, electron, and intravital microscopy, we showed that these contacts remarkably resembled immune synapses or kinapses, albeit with the absence of potential T cell receptor engagement. Th17 cells induced severe, localized, and partially reversible fluctuation in neuronal intracellular Ca
2+ concentration as an early sign of neuronal damage. These results highlight the central role of the Th17 cell effector phenotype for neuronal dysfunction in chronic neuroinflammation.
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► Th17 cells establish immune-neuronal synapses and induce neuronal cell death in vitro ► Th17 cells directly contact neurons irrespective of their CNS-antigen specificity ► Th17 cell-neuronal interaction leads to severe neuronal dysfunction ► Th17 cell-mediated Ca
2+ elevation is partially reversible by blocking excitotoxicity
Granulomatosis or eosinophilic granulomatosis with polyangiitis (GPA/EGPA) can affect multiple organs resulting in heterogeneous symptoms and phenotypes. Pituitary gland dysfunction rarely occurs in ...GPA (1–3%) and even less in EGPA (two case reports). Here, we report a case of a 51-year-old female patient with a four-year history of EGPA who presented with new polydipsia and polyuria. Laboratory testing and magnetic resonance imaging (MRI) confirmed pituitary gland dysfunction caused by a hypophysitis. Therapeutic adjustment with a switch from dupilumab to mepolizumab resulted in a decrease in clinical symptoms, inflammation in MRI, and normalization of C-reactive protein in serum. This case underlines hypophysitis as a rare organ involvement also in EGPA. Moreover, this case demonstrates the responsiveness of neuroinflammatory manifestations to the recently approved anti-interleukin-5 monoclonal antibody mepolizumab as a new potential treatment option.
Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the CNS. Myelin-specific CD4
Th lymphocytes are known to play a major role in both MS and its animal model ...experimental autoimmune encephalomyelitis (EAE). CCR7 is a critical element for immune cell trafficking and recirculation, that is, lymph node homing, under homeostatic conditions; blocking CCR7
central memory cells from egress of lymph nodes is a therapeutic approach in MS. To define the effect of CD4
T cell-specific constitutive deletion of CCR7 in the priming and effector phase in EAE, we used an active EAE approach in T cell reconstituted Rag1
mice, as well as adoptive transfer EAE, in which mice received in vitro-primed CCR7
or CCR7
myelin Ag TCR-transgenic 2d2 Th17 cells. Two-photon laser scanning microscopy was applied in living anesthetized mice to monitor the trafficking of CCR7-deficient and wild-type CD4
T cells in inflammatory lesions within the CNS. We demonstrate that CD4
T cell-specific constitutive deletion of CCR7 led to impaired induction of active EAE. In adoptive transfer EAE, mice receiving in vitro-primed CCR7
2d2 Th17 cells showed similar disease onset as mice adoptively transferred with CCR7
2d2 Th17 cells. Using two-photon laser scanning microscopy CCR7
and CCR7
CD4
T cells did not reveal differences in motility in either animal model of MS. These findings indicate a crucial role of CCR7 in neuroinflammation during the priming of autoimmune CD4
T cells but not in the CNS.
Abstract
Objective
The diagnosis of neurosarcoidosis (NS) remains challenging due to the difficulty to obtain central nervous system (CNS) biopsies. Various diagnostic parameters are considered for ...the definition of possible, probable and definite NS. Magnetic resonance imaging (MRI) is the imaging gold standard and considered in diagnostic criteria. Fluorodeoxyglucose positron emission (
18
F-FDG PET) is sometimes performed additionally to identify possible systemic biopsy targets. However, at present, its findings are not incorporated into the diagnostic criteria for neurosarcoidosis (NS).
Methods
We conducted a single center retrospective search for the period 2020–2022, for patients with neurological symptoms in a diagnostic context of suspected NS who underwent MRI and additional
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F-FDG PET scans to identify potential hypermetabolism in the CNS and biopsy targets.
Results
We identified three cases of NS, where Gadolinium-enhanced MRI scans did not show abnormalities while
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F-FDG PET revealed hypermetabolic lesions in areas of the CNS. Additional MRI scans were still inconclusive for structural changes. We diagnosed a “probable” NS in all cases with histopathological confirmation of systemic sarcoidosis which led to an intensified therapy regime.
Discussion
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F-FDG PET is an early indicator for metabolic changes. It appears to be a useful add-on to improve accuracy of diagnostic criteria in suspected NS without MRI findings.
Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in ...living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm(2)) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm(2)) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal dysfunction in neuroinflammation.
Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication ...both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.
The proneurotrophin receptor sortilin is a protein with dual functions, being involved in intracellular protein transport, as well as cellular signal transduction. The relevance of the receptor for ...various neuronal disorders, such as dementia, seizures, and brain injury, is well established. In contrast, little is known about the role of sortilin in immune cells and inflammatory diseases. The aim of our study was to elucidate the distribution of sortilin in different immune cell types in mice and humans and to analyze its function in autoimmune CNS inflammation. Sortilin was expressed most profoundly in murine and human macrophages and dendritic cells and to a much lesser extent in B and T cells. In dendritic cells, sortilin had an impact on Ag processing. Accordingly, sortilin was highly expressed by infiltrated perivascular myeloid cells, mainly in vessel cuffs, in the CNS of patients suffering from multiple sclerosis, the most common inflammatory autoimmune disease of the CNS. Yet, sortilin gene-targeted mice (Sort1(-/-)) and chimeras deficient in sortilin in the immune system were as susceptible as wild-type littermates to T cell-dependent experimental autoimmune encephalomyelitis. Considering our results and recent data from other investigators, we conclude that the proneurotrophin receptor sortilin plays a role in innate, rather than in adaptive, immune processes and, thus, not in autoimmune neuroinflammation.
Infratentorial lesions have been assigned an equivalent weighting to supratentorial plaques in the new McDonald criteria for diagnosing multiple sclerosis. Moreover, their presence has been shown to ...have prognostic value for disability. However, their spatial distribution and impact on network damage is not well understood. As a preliminary step in this study, we mapped the overall infratentorial lesion pattern in relapsing-remitting multiple sclerosis patients (N = 317) using MRI, finding the pons (lesion density, 14.25/cm(3)) and peduncles (13.38/cm(3)) to be predilection sites for infratentorial lesions. Based on these results, 118 fiber bundles from 15 healthy controls and a subgroup of 23 patients showing lesions unilaterally at the predilection sites were compared using diffusion tensor imaging to analyze the impact of an isolated infratentorial lesion on the affected fiber tracts. Fractional anisotropy, mean diffusion as well as axial and radial diffusivity were investigated at the lesion site and along the entire fiber tract. Infratentorial lesions were found to have an impact on the fractional anisotropy and radial diffusivity not only at the lesion site itself but also along the entire affected fiber tract. As previously found in animal experiments, inflammatory attack in the posterior fossa in multiple sclerosis impacts the whole affected fiber tract. Here, this damaging effect, reflected by changes in diffusivity measures, was detected in vivo in multiple sclerosis patients in early stages of the disease, thus demonstrating the influence of a focal immune attack on more distant networks, and emphasizing the pathophysiological role of Wallerian degeneration in multiple sclerosis.
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