Analyzing nicotinamide adenine dinucleotide phosphate oxidase activation in aging and vascular amyloid pathology

In aging individuals, both protective as well as regulatory immune functions are declining, resulting in an increased susceptibility to infections as well as to autoimmunity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2-deficiency in immune cell subsets has been shown to be associated with aging. Using intravital marker-free NAD(P)H-fluorescence lifetime imaging, we have previously identified microglia/myeloid cells and astrocytes as main cellular sources of NADPH oxidase (NOX) activity in the CNS during neuroinflammation, due to an overactivation of NOX. The overactivated NOX enzymes catalyze the massive production of the highly reactive O−2, which initiates in a chain reaction the overproduction of diverse reactive oxygen species (ROS). Age-dependent oxidative distress levels in the brain and their cellular sources are not known. Furthermore, it is unclear whether in age- dependent diseases oxidative distress is initiated by overproduction of ROS or by a decrease in antioxidant capacity, subsequently leading to neurodegeneration in the CNS. Here, we compare the activation level of NOX enzymes in the cerebral cortex of young and aged mice as well as in a model of vascular amyloid pathology. Despite the fact that a striking change in the morphology of microglia can be detected between young and aged individuals, we find comparable low-level NOX activation both in young and old mice. In contrast, aged mice with the human APPE693Q mutation, a model for cerebral amyloid angiopathy (CAA), displayed increased focal NOX overactivation in the brain cortex, especially in tissue areas around the vessels. Despite activated morphology in microglia, NOX overactivation was detected only in a small fraction of these cells, in contrast to other pathologies with overt inflammation as experimental autoimmune encephalomyelitis (EAE) or glioblastoma. Similar to these pathologies, the astrocytes majorly contribute to the NOX overactivation in the brain cortex during CAA. Together, these findings emphasize the role of other cellular sources of activated NOX than phagocytes not only during EAE but also in models of amyloid pathology. Moreover, they may strengthen the hypothesis that microglia/monocytes show a diminished potential for clearance of amyloid beta protein

Low-Density Granulocytes Are a Novel Immunopathological Feature in Both Multiple Sclerosis and Neuromyelitis Optica Spectrum Disorder

Objective: To investigate whether low-density granulocytes (LDGs) are an immunophenotypic feature of patients with multiple sclerosis (MS) or neuromyelitis optica spectrum disorder (NMOSD). Methods: Blood samples were collected from 20 patients with NMOSD and 17 patients with MS, as well as from 15 patients with Systemic Lupus Erythematosus (SLE) and 23 Healthy Donors (HD). We isolated peripheral blood mononuclear cells (PBMCs) with density gradient separation and stained the cells with antibodies against CD14, CD15, CD16, and CD45, and analyzed the cells by flow cytometry or imaging flow cytometry. We defined LDGs as CD14-CD15(high) and calculated their share in total PBMC leukocytes (CD45+) as well as the share of CD16(hi) LDGs. Clinical data on disease course, medication, and antibody status were obtained. Results: LDGs were significantly more common in MS and NMOSD than in HDs, comparable to SLE samples (median values HD 0.2%, MS 0.9%, NMOSD 2.1%, SLE 4.3%). 0/23 of the HDs, but 17/20 NMOSD and 11/17 MS samples as well as 13/15 SLE samples had at least 0.7 % LDGs. NMOSD patients without continuous immunosuppressive treatment had significantly more LDGs compared to their treated counterparts. LDG nuclear morphology ranged from segmented to rounded, suggesting a heterogeneity within the group. Conclusion: LDGs are a feature of the immunophenotype in some patients with MS and NMOSD

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

Multiplexed histology of COVID-19 post-mortem lung samples - PROLONGED CASE 1 FOV1

Image-based data set of a post-mortem lung sample from a COVID-19 donor (PROLONGED CASE 1 FOV1) Each image shows the same field of view (FOV), sequentially stained with the depicted fluorescence-labelled antibodies, including surface proteins, intracellular proteins and transcription factors. Images contain 2024 x 2024 pixels and are generated using an inverted wide-field fluorescence microscope with a 20x objective, a lateral resolution of 325 nm and an axial resolution above 5 µm. Images have been normalized and intensities adjusted.Funding: Deutsche Forschungsgemeinschaft HA5354/10-1, SPP1937 (HA5354/8-2) and TRR130 P17 and C01 (to Anja Hauser

Multiplexed histology of COVID-19 post-mortem lung samples - CONTROL CASE 2 FOV1

Image-based data set of a post-mortem lung sample from a non-COVID-19-related pneumonia donor (CONTROL CASE 2 FOV1) Each image shows the same field of view (FOV), sequentially stained with the depicted fluorescence-labelled antibodies, including surface proteins, intracellular proteins and transcription factors. Images contain 2024 x 2024 pixels and are generated using an inverted wide-field fluorescence microscope with a 20x objective, a lateral resolution of 325 nm and an axial resolution above 5 µm. Images have been normalized and intensities adjusted.Funding: Deutsche Forschungsgemeinschaft HA5354/10-1, SPP1937 (HA5354/8-2) and TRR130 P17 and C01 (to Anja Hauser

Multiplexed histology of COVID-19 post-mortem lung samples - ACUTE CASE 2 FOV1

Image-based data set of a post-mortem lung sample from a COVID-19 donor (ACUTE CASE 2 FOV1) Each image shows the same field of view (FOV), sequentially stained with the depicted fluorescence-labelled antibodies, including surface proteins, intracellular proteins and transcription factors. Images contain 2024 x 2024 pixels and are generated using an inverted wide-field fluorescence microscope with a 20x objective, a lateral resolution of 325 nm and an axial resolution above 5 µm. Images have been normalized and intensities adjusted.Funding: Deutsche Forschungsgemeinschaft HA5354/10-1, SPP1937 (HA5354/8-2) and TRR130 P17 and C01 (to Anja Hauser

Multiplexed histology of COVID-19 post-mortem lung samples - ACUTE CASE 2 FOV4

Image-based data set of a post-mortem lung sample from a COVID-19 donor (ACUTE CASE 2 FOV4) Each image shows the same field of view (FOV), sequentially stained with the depicted fluorescence-labelled antibodies, including surface proteins, intracellular proteins and transcription factors. Images contain 2024 x 2024 pixels and are generated using an inverted wide-field fluorescence microscope with a 20x objective, a lateral resolution of 325 nm and an axial resolution above 5 µm. Images have been normalized and intensities adjusted.Funding: Deutsche Forschungsgemeinschaft HA5354/10-1, SPP1937 (HA5354/8-2) and TRR130 P17 and C01 (to Anja Hauser

Multiplexed histology of COVID-19 post-mortem lung samples - ACUTE CASE 2 FOV3

Image-based data set of a post-mortem lung sample from a COVID-19 donor (ACUTE CASE 2 FOV3) Each image shows the same field of view (FOV), sequentially stained with the depicted fluorescence-labelled antibodies, including surface proteins, intracellular proteins and transcription factors. Images contain 2024 x 2024 pixels and are generated using an inverted wide-field fluorescence microscope with a 20x objective, a lateral resolution of 325 nm and an axial resolution above 5 µm. Images have been normalized and intensities adjusted.Funding: Deutsche Forschungsgemeinschaft HA5354/10-1, SPP1937 (HA5354/8-2) and TRR130 P17 and C01 (to Anja Hauser