Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry

The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically ...

Western University

University of Calgary

There is mounting scientific evidence showing the importance of innate biological rhythms on disease onset and progression. Perhaps the most important of these is the circadian rhythm, a cycle of oscillations lasting approximately 24 h. Recent work has shown that circadian rhythms are intrinsically linked to the immune system in a bidirectional fashion, and that disruption of these cycles can contribute to changes in pathology and quality of life (including fatigue, mood, and disability). This is particularly true in diseases of the nervous and immune systems. We review here the current preclinical and clinical literature to highlight interactions between circadian rhythms and multiple sclerosis, as well as its animal model, experimental autoimmune encephalomyelitis. We highlight potential benefits of chronotherapy (the temporal administration of immunomodulatory drugs) in an effort to increase treatment efficacy and reduce the negative side-effects of the drugs that often burden those suffering from the disease.

Chronobiological regulation of psychosocial and physiological outcomes in multiple sclerosis.

B cells represent a relatively minor cell population within both the healthy and diseased central nervous system (CNS), yet they can have profound effects. This is emphasized in multiple sclerosis, in which B cell-depleting therapies are arguably the most efficacious treatment for the condition. In this Review, we discuss how B cells enter and persist in the CNS and how, in many neurological conditions, B cells concentrate within CNS barriers but are rarely found in the parenchyma. We highlight how B cells can contribute to CNS pathology through antibody secretion, antigen presentation and secretion of neurotoxic molecules, using examples from CNS tumours, CNS infections and autoimmune conditions such as neuromyelitis optica and, in particular, multiple sclerosis. Overall, understanding common and divergent principles of B cell accumulation and their effects within the CNS could offer new insights into treating these devastating neurological conditions.

B cells in central nervous system disease: diversity, locations and pathophysiology.

Oxidative stress promotes tissue injury in the central nervous system in neurological disorders such as multiple sclerosis (MS). To protect against this, antioxidant enzymes including superoxide dismutase-1 (SOD1), heme oxygenase-1 (HO-1), peroxiredoxin-5 (PRDX5) and glutathione peroxidase-4 (GPX4) may be upregulated. However, whether antioxidant enzyme elevation in mouse models of neurodegeneration corresponds to their expression in human diseases such as MS requires investigation. Here, we analyzed and compared the expression of SOD1, HO-1, PRDX5 and GPX4 in the murine spinal cord of three models of MS: focal lesions induced by (1) oxidized phosphatidylcholine or (2) lysophosphatidylcholine (lysolecithin), and (3) diffuse lesions of experimental autoimmune encephalomyelitis. Notably, CD68 microglia/macrophages were the predominant cellular populations that expressed the highest levels of the detected antioxidant enzymes. Overall, the expression patterns of antioxidant enzymes across the models were similar. The increase of these antioxidant enzymes was corroborated in MS brain tissue using spatial RNA sequencing. Collectively, these results show that antioxidant capacity is relatively conserved between mouse models and MS lesions, and suggest a need to investigate whether the antioxidant elevation in microglia/macrophages is a protective response during oxidative injury, neurodegeneration, and MS.

Expression of antioxidant enzymes in lesions of multiple sclerosis and its models.

Microglia are the immune sentinels of the central nervous system with protective roles such as the removal of neurotoxic oxidized phosphatidylcholines (OxPCs). As aging alters microglial function and elevates neurological disability in diseases such as multiple sclerosis, defining aging-associated factors that cause microglia to lose their custodial properties or even become injurious can help to restore their homeostasis. We used single-cell and spatial RNA sequencing in the spinal cord of young (6-week-old) and middle-aged (52-week-old) mice to determine aging-driven microglial reprogramming at homeostasis or after OxPC injury. We identified numerous aging-associated microglial transcripts including osteopontin elevated in OxPC-treated 52-week-old mice, which correlated with greater neurodegeneration. Osteopontin delivery into the spinal cords of 6-week-old mice worsened OxPC lesions, while its knockdown in 52-week-old lesions attenuated microglial inflammation and axon loss. Thus, elevation of osteopontin and other transcripts in aging disorders including multiple sclerosis perturbs microglial functions contributing to aging-associated neurodegeneration.

Single-cell and spatial RNA sequencing identify perturbators of microglial functions with aging.

Immunofluorescence histology is commonly used to study immune cells in tissues where the number of fluorescence parameters is normally limited to four or less. This makes it impossible to interrogate multiple subsets of immune cells in tissue with the same precision as flow cytometry. The latter, however, dissociates tissues and loses spatial information. To bridge the gap between these technologies, we developed a workflow to expand the number of fluorescence parameters that can be imaged on widely available microscopes. We instituted a method for identifying single cells in tissue and exporting the data for flow cytometry-based analysis. This histoflow cytometry technique successfully separates spectrally overlapping dyes and identifies similar numbers of cells in tissue sections as manual cell counts. Populations identified through flow cytometry-like gating strategies are mapped to the original tissue to spatially localize gated subsets. We applied histoflow cytometry to immune cells in the spinal cords of mice with experimental autoimmune encephalomyelitis. We ascertained that B cells, T cells, neutrophils, and phagocytes differed in their frequencies in CNS immune cell infiltrates and were increased relative to healthy controls. Spatial analysis determined that B cells and T cells/phagocytes preferentially localized to CNS barriers and parenchyma, respectively. By spatially mapping these immune cells, we inferred their preferred interacting partners within immune cell clusters. Overall, we demonstrate the ease and utility of histoflow cytometry, which expands the number of fluorescent channels used in conventional immunofluorescence and enables quantitative cytometry and spatial localization of histological analyses.

Single Cell Analysis of High-Parameter Histology Images Using Histoflow Cytometry.

The extracellular matrix (ECM) of the central nervous system (CNS) is an interconnected network of proteins and sugars with critical roles in both homeostasis and disease. In neurological diseases, excessive ECM deposition and remodeling impact both injury and repair. CNS lesions of multiple sclerosis (MS), a chronic inflammatory and degenerative disease, cause prominent alterations of the ECM. However, there are a lack of data investigating how the multitude of ECM members change in relation to each other and how this affects the MS disease course. Here, we evaluated ECM changes in MS lesions compared to a control brain using databases generated in-house through spatial mRNA-sequencing and through a public resource of single-nucleus RNA sequencing previously published by Absinta and colleagues. These results underline the importance of publicly available datasets to find new targets of interest, such as the ECM. Both spatial and public datasets demonstrated widespread changes in ECM molecules and their interacting proteins, including alterations to proteoglycans and glycoproteins within MS lesions. Some of the altered ECM members have been described in MS, but other highly upregulated members, including the SPARC family of proteins, have not previously been highlighted. SPARC family members are upregulated in other conditions by reactive astrocytes and may influence immune cell activation and MS disease course. The profound changes to the ECM in MS lesions deserve more scrutiny as they impact neuroinflammation, injury, and repair.

Rajiv W. Jain

Department of Microbiology and Immunology

Western University

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry

Rajiv W. Jain

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Western University

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry

Department of Microbiology and Immunology