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

The chronically inflamed central nervous system provides niches for long-lived plasma cells

Abstract Although oligoclonal bands in the cerebrospinal fluid have been a hallmark of multiple sclerosis diagnosis for over three decades, the role of antibody-secreting cells in multiple sclerosis remains unclear. T and B cells are critical for multiple sclerosis pathogenesis, but increasing evidence suggests that plasma cells also contribute, through secretion of autoantibodies. Long-lived plasma cells are known to drive various chronic inflammatory conditions as e.g. systemic lupus erythematosus, however, to what extent they are present in autoimmune central nervous system inflammation has not yet been investigated. In brain biopsies from multiple sclerosis patients and other neurological diseases, we could detect non-proliferating plasma cells (CD138+Ki67−) in the parenchyma. Based on this finding, we hypothesized that long-lived plasma cells can persist in the central nervous system (CNS). In order to test this hypothesis, we adapted the multiple sclerosis mouse model experimental autoimmune encephalomyelitis to generate a B cell memory response. Plasma cells were found in the meninges and the parenchyma of the inflamed spinal cord, surrounded by tissue areas resembling survival niches for these cells, characterized by an up-regulation of chemokines (CXCL12), adhesion molecules (VCAM-1) and survival factors (APRIL and BAFF). In order to determine the lifetime of plasma cells in the chronically inflamed CNS, we labeled the DNA of proliferating cells with 5-ethynyl-2′-deoxyuridine (EdU). Up to five weeks later, we could detect EdU+ long-lived plasma cells in the murine CNS. To our knowledge, this is the first study describing non-proliferating plasma cells directly in the target tissue of a chronic inflammation in humans, as well as the first evidence demonstrating the ability of plasma cells to persist in the CNS, and the ability of the chronically inflamed CNS tissue to promote this persistence. Hence, our results suggest that the CNS provides survival niches for long-lived plasma cells, similar to the niches found in other organs. Targeting these cells in the CNS offers new perspectives for treatment of chronic autoimmune neuroinflammatory diseases, especially in patients who do not respond to conventional therapies

Additional file 1: Figure S1. of The chronically inflamed central nervous system provides niches for long-lived plasma cells

Non-proliferating CD138+ cells in the brain of patients with other inflammatory neurological diseases (OND). DAPI (blue), CD138 (green) and Ki67 (red) were stained in the CNS of patient biopsies with other neurological diseases (OND, n = 4) as indicated on the left. Representative images are shown. White arrows indicate Ki67+ CD138+ cells. Scale bars represent 50 μm. (TIFF 14779 kb

Additional file 4: of The chronically inflamed central nervous system provides niches for long-lived plasma cells

Supplementary methods. (PDF 133 kb

Additional file 5: Figure S4. of The chronically inflamed central nervous system provides niches for long-lived plasma cells

BAFF-positive B cells and plasma cells in the inflamed CNS. Mice were immunized and boosted (day 28) with rhMOG. Analysis of spinal cord was performed during peak after boost. The fluorescence signal of DAPI (blue), BAFF (red), kappa/lambda (κ/λ, upper panel green) and B220 (lower panel green) is shown. A plasma cell is indicated with an arrow. Three mice of two independent experiments were analyzed. Scale bars represent 50 μm. (TIFF 20356 kb

High-Resolution Intravital Microscopy

<div><p>Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy - the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector.</p> </div

Comparison of multi-beam striped-illumination TPLSM to standard single- and multi-beam TPLSM.

<p>(<b>a</b>) 3D fluorescence images of spleen slices of chimera EGFP mice reconstituted with a non-fluorescent immune system using single-beam scanning PMT-TPLSM (SB-PMT), multi-beam scanning CCD-TPLM (MB-CCD) and SI-TPLSM (MB-SI). λ_exc = 920 nm, grid unit = 16.5 µm, grid unit of the cropped image = 2.0 µm. (<b>b</b>) 3D fluorescence images of the same regions within a popliteal lymph node after immunization with NP-CGG as recorded by MB-CCD-TPLSM versus MB-SI-TPLSM or by SB-PMT-TPLSM versus MB-SI-TPLSM. Follicular dendritic cells (FDC) are stained by CD21/CD35-Fab fragment-ATTO590 (magenta), while antigen-specific B1–8 cells express EGFP (green). λ_exc = 860 nm, grid unit = 15 µm. (<b>c</b>) 3D second-harmonic generation (SHG) signal images of the same region within a non-fluorescent lymph node as recorded by MB-CCD-, SB-PMT- and MB-SI-TPLSM. The SHG signal mainly originates from collagen fibers. λ_exc = 900 nm, grid unit = 10 µm (grid unit = 8 µm for MB-CCD-, MB-SI-TPLSM and 6 µm for SB-PMT-TPLSM). (<b>d</b>) 3D fluorescence images of acute cerebellum slices of CerTN L15 mice (expresses Cerulean and Citrine over the Thy1 cassette) recorded with the same set-ups. λ_exc = 850 nm, grid unit = 20 µm. All experiments were performed with the 20×, NA = 0.95 objective lens at <i>z</i>-step = 500 nm.</p

Lateral and axial resolutions measured with the 20× and 40× magnification objective lenses, respectively, on fluorescent microbeads or, for second harmonic generation, on collagen fibers within lymph nodes.

<p>Lateral and axial resolutions measured with the 20× and 40× magnification objective lenses, respectively, on fluorescent microbeads or, for second harmonic generation, on collagen fibers within lymph nodes.</p