P2R Inhibitors Prevent Antibody-Mediated Complement Activation in an Animal Model of Neuromyelitis Optica : P2R Inhibitors Prevent Autoantibody Injury.

Purinergic 2 receptors (P2Rs) contribute to disease-related immune cell signaling and are upregulated in various pathological settings, including neuroinflammation. P2R inhibitors have been used to treat inflammatory diseases and can protect against complement-mediated cell injury. However, the mechanisms behind these anti-inflammatory properties of P2R inhibitors are not well understood, and their potential in CNS autoimmunity is underexplored. Here, we tested the effects of P2R inhibitors on glial toxicity in a mouse model of neuromyelitis optica spectrum disorder (NMOSD). NMOSD is a destructive CNS autoimmune disorder, in which autoantibodies against astrocytic surface antigen Aquaporin 4 (AQP4) mediate complement-dependent loss of astrocytes. Using two-photon microscopy in vivo, we found that various classes of P2R inhibitors prevented AQP4-IgG/complement-dependent astrocyte death. In vitro, these drugs inhibited the binding of AQP4-IgG or MOG-IgG to their antigen in a dose-dependent manner. Size-exclusion chromatography and circular dichroism spectroscopy revealed a partial unfolding of antibodies in the presence of various P2R inhibitors, suggesting a shared interference with IgG antibodies leading to their conformational change. Our study demonstrates that P2R inhibitors can disrupt complement activation by direct interaction with IgG. This mechanism is likely to influence the role of P2R inhibitors in autoimmune disease models and their therapeutic impact in human disease.

A new form of axonal pathology in a spinal model of neuromyelitis optica.

Neuromyelitis optica is a chronic neuroinflammatory disease, which primarily targets astrocytes and often results in severe axon injury of unknown mechanism. Neuromyelitis optica patients harbour autoantibodies against the astrocytic water channel protein, aquaporin-4 (AQP4-IgG), which induce complement-mediated astrocyte lysis and subsequent axon damage. Using spinal in vivo imaging in a mouse model of such astrocytopathic lesions, we explored the mechanism underlying neuromyelitis optica-related axon injury. Many axons showed a swift and morphologically distinct 'pearls-on-string' transformation also readily detectable in human neuromyelitis optica lesions, which especially affected small calibre axons independently of myelination. Functional imaging revealed that calcium homeostasis was initially preserved in this 'acute axonal beading' state, ruling out disruption of the axonal membrane, which sets this form of axon injury apart from previously described forms of traumatic and inflammatory axon damage. Morphological, pharmacological and genetic analyses showed that AQP4-IgG-induced axon injury involved osmotic stress and ionic overload, but does not appear to use canonical pathways of Wallerian-like degeneration. Subcellular analysis demonstrated remodelling of the axonal cytoskeleton in beaded axons, especially local loss of microtubules. Treatment with the microtubule stabilizer epothilone, a putative therapy approach for traumatic and degenerative axonopathies, prevented axonal beading, while destabilizing microtubules sensitized axons for beading. Our results reveal a distinct form of immune-mediated axon pathology in neuromyelitis optica that mechanistically differs from known cascades of post-traumatic and inflammatory axon loss, and suggest a new strategy for neuroprotection in neuromyelitis optica and related diseases.

Skin and gut imprinted helper T cell subsets exhibit distinct functional phenotypes in central nervous system autoimmunity.

Multidimensional single-cell analyses of T cells have fueled the debate about whether there is extensive plasticity or 'mixed' priming of helper T cell subsets in vivo. Here, we developed an experimental framework to probe the idea that the site of priming in the systemic immune compartment is a determinant of helper T cell-induced immunopathology in remote organs. By site-specific in vivo labeling of antigen-specific T cells in inguinal (i) or gut draining mesenteric (m) lymph nodes, we show that i-T cells and m-T cells isolated from the inflamed central nervous system (CNS) in a model of multiple sclerosis (MS) are distinct. i-T cells were Cxcr6+, and m-T cells expressed P2rx7. Notably, m-T cells infiltrated white matter, while i-T cells were also recruited to gray matter. Therefore, we propose that the definition of helper T cell subsets by their site of priming may guide an advanced understanding of helper T cell biology in health and disease.

In vivo imaging reveals rapid astrocyte depletion and axon damage in a model of neuromyelitis optica-related pathology.

OBJECTIVE: Neuromyelitis optica (NMO) is an autoimmune disease of the central nervous system, which resembles multiple sclerosis (MS). NMO differs from MS, however, in the distribution and histology of neuroinflammatory lesions and shows a more aggressive clinical course. Moreover, the majority of NMO patients carry immunoglobulin G autoantibodies against aquaporin-4 (AQP4), an astrocytic water channel. Antibodies against AQP4 can damage astrocytes by complement, but NMO histopathology also shows demyelination, and - importantly-axon injury, which may determine permanent deficits following NMO relapses. The dynamics of astrocyte injury in NMO and the mechanisms by which toxicity spreads to axons are not understood. METHODS: Here, we establish in vivo imaging of the spinal cord, one of the main sites of NMO pathology, as a powerful tool to study the formation of experimental NMO-related lesions caused by human AQP4 antibodies in mice. RESULTS: We found that human AQP4 antibodies caused acute astrocyte depletion with initial oligodendrocyte survival. Within 2 hours of antibody application, we observed secondary axon injury in the form of progressive swellings. Astrocyte toxicity and axon damage were dependent on AQP4 antibody titer and complement, specifically C1q. INTERPRETATION: In vivo imaging of the spinal cord reveals the swift development of NMO-related acute axon injury after AQP4 antibody-mediated astrocyte depletion. This approach will be useful in studying the mechanisms underlying the spread of NMO pathology beyond astrocytes, as well as in evaluating potential neuroprotective interventions. Ann Neurol 2016;79:794-805.