Th2 and metabolic responses to nematodes are independent of prolonged host microbiota abrogation.

Antibiotic treatment can lead to elimination of both pathogenic bacteria and beneficial commensals, as well as to altered host immune responses. Here, we investigated the influence of prolonged antibiotic treatment (Abx) on effector, memory and recall Th2 immune responses during the primary infection, memory phase and secondary infection with the small intestinal nematode Heligmosomoides polygyrus. Abx treatment significantly reduced gut bacterial loads, but neither worm burdens, nor worm fecundity in primary infection were affected, only worm burdens in secondary infection were elevated in Abx treated mice. Abx mice displayed trends for elevated effector and memory Th2 responses during primary infection, but overall frequencies of Th2 cells in the siLP, PEC, mLN and in the spleen were similar between Abx treated and untreated groups. Gata3+ effector and memory Th2 cytokine responses also remained unimpaired by prolonged Abx treatment. Similarly, the energy production and defence mechanisms of the host tissue and the parasite depicted by NAD(P)H fluorescence lifetime imaging (FLIM) did not change by the prolonged use of antibiotics. We show evidence that the host Th2 response to intestinal nematodes, as well as host and parasite metabolic pathways are robust and remain unimpaired by host microbiota abrogation.

Two-Photon Excitation Spectra of Various Fluorescent Proteins within a Broad Excitation Range.

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720.

Teriflunomide Preserves Neuronal Activity and Protects Mitochondria in Brain Slices Exposed to Oxidative Stress.

Teriflunomide (TFN) limits relapses in relapsing-remitting multiple sclerosis (RRMS) by reducing lymphocytic proliferation through the inhibition of the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) and the subsequent modulation of de novo pyrimidine synthesis. Alterations of mitochondrial function as a consequence of oxidative stress have been reported during neuroinflammation. Previously, we showed that TFN prevents alterations of mitochondrial motility caused by oxidative stress in peripheral axons. Here, we aimed to validate TFN effects on mitochondria and neuronal activity in hippocampal brain slices, in which cellular distribution and synaptic circuits are largely preserved. TFN effects on metabolism and neuronal activity were investigated by assessing oxygen partial pressure and local field potential in acute slices. Additionally, we imaged mitochondria in brain slices from the transgenic Thy1-CFP/COX8A)S2Lich/J (mitoCFP) mice using two-photon microscopy. Although TFN could not prevent oxidative stress-related depletion of ATP, it preserved oxygen consumption and neuronal activity in CNS tissue during oxidative stress. Furthermore, TFN prevented mitochondrial shortening and fragmentation of puncta-shaped and network mitochondria during oxidative stress. Regarding motility, TFN accentuated the decrease in mitochondrial displacement and increase in speed observed during oxidative stress. Importantly, these effects were not associated with neuronal viability and did not lead to axonal damage. In conclusion, during conditions of oxidative stress, TFN preserves the functionality of neurons and prevents morphological and motility alterations of mitochondria.

[The DRFZ-a pioneer in research on the interaction between immune and stromal cells during de- and regeneration of the musculoskeletal system]. / Das DRFZ ­ ein Vorreiter bei der Erforschung des Zusammenspiels von Immun- und Stromazellen bei der De- und Regeneration des Bewegungsapparats.

Rheumatoid arthritis and osteoarthritis are two related chronic diseases of the musculoskeletal system which are particularly pronounced in the region of joints and bones. Their pathogeneses are associated with chronic inflammation, which can disrupt homeostasis in bones and articular cartilage. Degradation products deriving from articular cartilage can contribute to the exacerbation of inflammation in the joint region. Mechanical stimuli and blood vessels also play a central role in both the regulation of bone growth as well as in the regeneration of bone tissue. Not only chronic inflammatory processes but also hormonal changes after menopause or undesired effects of glucocorticoid therapy have an influence on the balance between bone resorption and deposition, by promoting the former and reducing the latter. This results in decreased bone quality and, in some cases, considerable loss of bone or osteoporosis. An in-depth understanding of these processes at the molecular, cellular, and tissue level, as well as of the changes present in chronic inflammatory diseases, has been the focus of research at the German Rheumatism Research Center (Deutsches Rheuma-Forschungszentrum, DRFZ) since its foundation. Based on an improved understanding of these mechanisms, the DRFZ aims to develop improved prevention and treatment strategies with effects even in early disease stages.

Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix.

Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach.

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies.

Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Coregistered Spectral Optical Coherence Tomography and Two-Photon Microscopy for Multimodal Near-Instantaneous Deep-Tissue Imaging.

Two-photon microscopy (2PM) has brought unique insight into the mechanisms underlying immune system dynamics and function since it enables monitoring of cellular motility and communication in complex systems within their genuine environment-the living organism. However, use of 2PM in clinical settings is limited. In contrast, optical coherence tomography (OCT), a noninvasive label-free diagnostic imaging method, which allows monitoring morphologic changes of large tissue regions in vivo, has found broad application in the clinic. Here we developed a combined multimodal technology to achieve near-instantaneous coregistered OCT, 2PM, and second harmonic generation (SHG) imaging over large volumes (up to 1,000 × 1,000 × 300 µm3 ) of tendons and other tissue compartments in mouse paws, as well as in mouse lymph nodes, spleens, and femurs. Using our multimodal imaging approach, we found differences in macrophage cell shape and motility behavior depending on whether they are located in tendons or in the surrounding tissue compartments of the mouse paw. The cellular shape of tissue-resident macrophages, indicative for their role in tissue, correlated with the supramolecular organization of collagen as revealed by SHG and OCT. Hence, the here-presented approach of coregistered OCT and 2PM has the potential to link specific cellular phenotypes and functions (as revealed by 2PM) to tissue morphology (as highlighted by OCT) and thus, to build a bridge between basic research knowledge and clinical observations. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

In vivo imaging of partially reversible th17 cell-induced neuronal dysfunction in the course of encephalomyelitis.

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.

Systematic Enzyme Mapping of Cellular Metabolism by Phasor-Analyzed Label-Free NAD(P)H Fluorescence Lifetime Imaging.

In the past years, cellular metabolism of the immune system experienced a revival, as it has become clear that it is not merely responsible for the cellular energy supply, but also impacts on many signaling pathways and, thus, on diverse cellular functions. Label-free fluorescence lifetime imaging of the ubiquitous coenzymes NADH and NADPH (NAD(P)H-FLIM) makes it possible to monitor cellular metabolism in living cells and tissues and has already been applied to study metabolic changes both under physiologic and pathologic conditions. However, due to the complex distribution of NAD(P)H-dependent enzymes in cells, whose distribution continuously changes over time, a thorough interpretation of NAD(P)H-FLIM results, in particular, resolving the contribution of various enzymes to the overall metabolic activity, remains challenging. We developed a systematic framework based on angle similarities of the phase vectors and their length to analyze NAD(P)H-FLIM data of cells and tissues based on a generally valid reference system of highly abundant NAD(P)H-dependent enzymes in cells. By using our analysis framework, we retrieve information not only about the overall metabolic activity, i.e., the fraction of free to enzyme-bound NAD(P)H, but also identified the enzymes predominantly active within the sample at a certain time point with subcellular resolution. We verified the performance of the approach by applying NAD(P)H-FLIM on a stromal-like cell line and identified a different group of enzymes that were active in the cell nuclei as compared to the cytoplasm. As the systematic phasor-based analysis framework of label-free NAD(P)H-FLIM can be applied both in vitro and in vivo, it retains the unique power to enable dynamic enzyme-based metabolic investigations, at subcellular resolution, in genuine environments.

Multiplexed fluorescence microscopy reveals heterogeneity among stromal cells in mouse bone marrow sections.

The bone marrow (BM) consists of multiple, structured micro-environmental entities-the so called niches, which contain hematopoietic cells as well as stromal cells. These niches fulfill a variety of functions, such as control of the hematopoietic stem cell pool, differentiation of hematopoietic cells, and maintenance of immunological memory. However, due to the molecular and cellular complexity and a lack of suitable histological multiplexing methods, the composition of the various BM niches is still elusive. In this study, we apply multiepitope-ligand-cartography (MELC) on bone sections from mice. We combine multiplexed immunofluorescence histology data with various object-based segmentation approaches in order to define irregularly shaped, net-like structures of stromal cells. We confirm MELC as a robust histological method and validate our automated segmentation algorithms using flow cytometry and manual evaluation. By means of MELC multiplexing, we reveal heterogeneous expression of leptin receptor (LpR), BP-1, and VCAM-1 in the stromal network. Moreover, we demonstrate by quantification a preferential contact of B cell subsets as well as of plasma cells to processes of CXCL12-expressing stromal cells, compared with stromal somata. In summary, our approach is suitable for spatial analysis of complex tissue structures.

Cell shape characterization and classification with discrete Fourier transforms and self-organizing maps.

Cells in their natural environment often exhibit complex kinetic behavior and radical adjustments of their shapes. This enables them to accommodate to short- and long-term changes in their surroundings under physiological and pathological conditions. Intravital multi-photon microscopy is a powerful tool to record this complex behavior. Traditionally, cell behavior is characterized by tracking the cells' movements, which yields numerous parameters describing the spatiotemporal characteristics of cells. Cells can be classified according to their tracking behavior using all or a subset of these kinetic parameters. This categorization can be supported by the a priori knowledge of experts. While such an approach provides an excellent starting point for analyzing complex intravital imaging data, faster methods are required for automated and unbiased characterization. In addition to their kinetic behavior, the 3D shape of these cells also provide essential clues about the cells' status and functionality. New approaches that include the study of cell shapes as well may also allow the discovery of correlations amongst the track- and shape-describing parameters. In the current study, we examine the applicability of a set of Fourier components produced by Discrete Fourier Transform (DFT) as a tool for more efficient and less biased classification of complex cell shapes. By carrying out a number of 3D-to-2D projections of surface-rendered cells, the applied method reduces the more complex 3D shape characterization to a series of 2D DFTs. The resulting shape factors are used to train a Self-Organizing Map (SOM), which provides an unbiased estimate for the best clustering of the data, thereby characterizing groups of cells according to their shape. We propose and demonstrate that such shape characterization is a powerful addition to, or a replacement for kinetic analysis. This would make it especially useful in situations where live kinetic imaging is less practical or not possible at all. © 2017 International Society for Advancement of Cytometry.

Disturbed canonical nuclear factor of κ light chain signaling in B cells of patients with common variable immunodeficiency.

BACKGROUND: Most patients with common variable immunodeficiency (CVID) present with severely reduced switched memory B-cell counts, and some display an increase of CD21low B-cell counts (CVID 21low), whereas others do not (CVID 21norm). Altered B-cell receptor (BCR) signaling might contribute to the defective memory formation observed in patients with CVID. OBJECTIVE: We sought to investigate canonical nuclear factor of κ light chain (NF-κB) signaling in B cells from patients with CVID as a central pathway in B-cell differentiation. METHODS: Degradation of inhibitor of κBα (IκBα) and p65 phosphorylation, nuclear translocation of p65, and regulation of target genes and cell function were investigated after different modes of B-cell stimulation. RESULTS: BCR-mediated canonical NF-κB signaling was impaired in all mature naive CVID-derived B cells. This impairment was more profound in naive B cells from CVID 21low patients than CVID 21norm patients and most pronounced in CD21low B cells. The signaling defect translated into reduced induction of Bcl-xL and IκBα, 2 bona fide target genes of the canonical NF-κB pathway. CD40 ligand- and Toll-like receptor 9-mediated signaling were less strongly altered. Signaling in CD21low B cells but not CD21+ B cells of patients with HIV was similarly affected. CONCLUSION: Combined with the previous description of disturbed Ca2+ signaling, the discovery of NF-κB signaling defects, especially in CVID 21low patients, suggests a broad underlying signaling defect affecting especially BCR-derived signals. Given the immune phenotype of monogenic defects affecting Ca2+ and NF-κB signaling, the latter is more likely to contribute to the humoral deficiency. The strongly disturbed BCR signaling of CD21low B cells is characteristic for this cell type and independent of the underlying disease.

NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes.

The balance between various cellular subsets of the innate and adaptive immune system and microbiota in the gastrointestinal tract is carefully regulated to maintain tolerance to the normal flora and dietary antigens, while protecting against pathogens. The intestinal epithelial cells and the network of dendritic cells and macrophages in the lamina propria are crucial lines of defense that regulate this balance. The complex relationship between the myeloid compartment (dendritic cells and macrophages) and lymphocyte compartment (T cells and innate lymphoid cells), as well as the impact of the epithelial cell layer have been studied in depth in recent years, revealing that the regulatory and effector functions of both innate and adaptive immune compartments exhibit more plasticity than had been previously appreciated. However, little is known about the metabolic activity of these cellular compartments, which is the basic function underlying all other additional tasks the cells perform. Here we perform intravital NAD(P)H fluorescence lifetime imaging in the small intestine of fluorescent reporter mice to monitor the NAD(P)H-dependent metabolism of epithelial and myeloid cells. The majority of myeloid cells which comprise the surveilling network in the lamina propria have a low metabolic activity and remain resting even upon stimulation. Only a few myeloid cells, typically localized at the tip of the villi, are metabolically active and are able to activate NADPH oxidases upon stimulation, leading to an oxidative burst. In contrast, the epithelial cells are metabolically highly active and, although not considered professional phagocytes, are also able to activate NADPH oxidases, leading to massive production of reactive oxygen species. Whereas the oxidative burst in myeloid cells is mainly catalyzed by the NOX2 isotype, in epithelial cells other isotypes of the NADPH oxidases family are involved, especially NOX4. They are constitutively expressed by the epithelial cells, but activated only on demand to ensure rapid defense against pathogens. This minimizes the potential for inadvertent damage from resting NOX activation, while maintaining the capacity to respond quickly if needed.

Phasor-Based Endogenous NAD(P)H Fluorescence Lifetime Imaging Unravels Specific Enzymatic Activity of Neutrophil Granulocytes Preceding NETosis.

Time-correlated single-photon counting combined with multi-photon laser scanning microscopy has proven to be a versatile tool to perform fluorescence lifetime imaging in biological samples and, thus, shed light on cellular functions, both in vitro and in vivo. Here, by means of phasor-analyzed endogenous NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence lifetime imaging, we visualize the shift in the cellular metabolism of healthy human neutrophil granulocytes during phagocytosis of Staphylococcus aureus pHrodo™ beads. We correlate this with the process of NETosis, i.e., trapping of pathogens by DNA networks. Hence, we are able to directly show the dynamics of NADPH oxidase activation and its requirement in triggering NETosis in contrast to other pathways of cell death and to decipher the dedicated spatio-temporal sequence between NADPH oxidase activation, nuclear membrane disintegration and DNA network formation. The endogenous FLIM approach presented here uniquely meets the increasing need in the field of immunology to monitor cellular metabolism as a basic mechanism of cellular and tissue functions.

Assessing Mitochondrial Movement Within Neurons: Manual Versus Automated Tracking Methods.

Owing to the small size of mitochondria and the complexity of their motility patterns, mitochondrial tracking is technically challenging. Mitochondria are often tracked manually; however, this is time-consuming and prone to measurement error. Here, we examined the suitability of four commercial and open-source software alternatives for automated mitochondrial tracking in neurons compared with manual measurements. We show that all the automated tracking tools dramatically underestimated track length, mitochondrial displacement and movement duration, with reductions ranging from 45 to 77% of the values obtained manually. In contrast, mitochondrial velocity was generally overestimated. Only the number of motile mitochondria and their directionality were similar between strategies. Despite these discrepancies, we show that automated tools successfully detected transport alterations after applying an oxidant agent. Thus, automated methods appear to be suitable for assessing relative transport differences between experimental groups, but not for absolute quantification of mitochondrial dynamics. Although useful for objective and time-efficient measurements of mitochondrial movements, results provided by automated methods should be interpreted with caution.

Quantitative Imaging of Ca2+ by 3D-FLIM in Live Tissues.

The calcium concentration within living cells is highly dynamic and, for many cell types, a reliable indicator of the functional state of the cells-both of isolated cells, but even, more important, of cells in tissue. In order to dynamically quantify intracellular calcium levels, various genetically encoded calcium sensors have been developed-the best of which are those based on Förster resonant energy transfer (FRET). Here we present a fluorescence lifetime imaging (FLIM) method to measure FRET in such a calcium sensor (TN L15) in neurons of hippocampal slices and of the brain stem of anesthetized mice. The method gives the unique opportunity to determine absolute neuronal calcium concentrations in the living organism.

Time lapse in vivo microscopy reveals distinct dynamics of microglia-tumor environment interactions-a new role for the tumor perivascular space as highway for trafficking microglia.

Microglial cells are critical for glioma growth and progression. However, only little is known about intratumoral microglial behavior and the dynamic interaction with the tumor. Currently the scarce understanding of microglial appearance in malignant gliomas merely originates from histological studies and in vitro investigations. In order to understand the pattern of microglia activity, motility and migration we designed an intravital study in an orthotopic murine glioma model using CX3CR1-eGFP(GFP/wt) mice. We analysed the dynamics of intratumoral microglia accumulation and activity, as well as microglia/tumor blood vessel interaction by epi-illumination and 2-photon laser scanning microscopy. We further investigated cellular and tissue function, including the enzyme activity of intratumoral and microglial NADPH oxidase measured by in vivo fluorescence lifetime imaging. We identified three morphological phenotypes of tumor-associated microglia cells with entirely different motility patterns. We found that NADPH oxidase activation is highly divergent in these microglia subtypes leading to different production levels of reactive oxygen species (ROS). We observed that microglia motility is highest within the perivascular niche, suggesting relevance of microglia/tumor blood vessel interactions. In line, reduction of tumor blood vessels by antivascular therapy confirmed the relevance of the tumor vessel compartment on microglia biology in brain tumors. In summary, we provide new insights into in vivo microglial behavior, regarding both morphology and function, in malignant gliomas. GLIA 2016;64:1210-1226.

Static and dynamic components synergize to form a stable survival niche for bone marrow plasma cells.

In the bone marrow (BM), memory plasma cells (PCs) survive for long time periods in dedicated microenvironmental survival niches, resting in terms of proliferation. Several cell types, such as eosinophils and reticular stromal cells, have been reported to contribute to the survival niche of memory PCs. However, until now it has not been demonstrated whether the niche is formed by a fixed cellular microenvironment. By intravital microscopy, we provide for the first time evidence that the direct contacts formed between PCs and reticular stromal cells are stable in vivo, and thus the PCs are sessile in their niches. The majority (∼ 80%) of PCs directly contact reticular stromal cells in a non-random fashion. The mesenchymal reticular stromal cells in contact with memory PCs are not proliferating. On the other hand, we show here that eosinophils in the vicinity of long-lived PCs are vigorously proliferating cells and represent a dynamic component of the survival niche. In contrast, if eosinophils are depleted by irradiation, newly generated eosinophils localize in the vicinity of radiation-resistant PCs and the stromal cells. These results suggest that memory PC niches may provide attraction for eosinophils to maintain stability with fluctuating yet essential accessory cells.

Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation.

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation.