Repetitive hypoxic preconditioning induces an immunosuppressed B cell phenotype during endogenous protection from stroke

Journal of Neuroinflammation (Impact Factor: 5.41). 01/2014; 11(1):22. DOI: 10.1186/1742-2094-11-22
Source: PubMed


Repetitive hypoxic preconditioning (RHP) creates an anti-inflammatory phenotype that protects from stroke-induced injury for months after a 2-week treatment. The mechanisms underlying long-term tolerance are unknown, though one exposure to hypoxia significantly increased peripheral B cell representation. For this study, we sought to determine if RHP specifically recruited B cells into the protected ischemic hemisphere, and whether RHP could phenotypically alter B cells prior to stroke onset.
Adult, male SW/ND4 mice received RHP (nine exposures over 2 weeks; 8 to 11 % O2; 2 to 4 hours) or identical exposures to 21 % O2 as control. Two weeks following RHP, a 60-minute transient middle cerebral artery occlusion was induced. Standard techniques quantified CXCL13 mRNA and protein expression. Two days after stroke, leukocytes were isolated from brain tissue (70:30 discontinuous Percoll gradient) and profiled on a BD-FACS Aria flow cytometer. In a separate cohort without stroke, sorted splenic CD19+ B cells were isolated 2 weeks after RHP and analyzed on an Illumina MouseWG-6 V2 Bead Chip. Final gene pathways were determined using Ingenuity Pathway Analysis. Student's t-test or one-way analysis of variance determined significance (P < 0.05).
CXCL13, a B cell-specific chemokine, was upregulated in post-stroke cortical vessels of both groups. In the ischemic hemisphere, RHP increased B cell representation by attenuating the diapedesis of monocyte, macrophage, neutrophil and T cells, to quantities indistinguishable from the uninjured, contralateral hemisphere. Pre-stroke splenic B cells isolated from RHP-treated mice had >1,900 genes differentially expressed by microarray analysis. Genes related to B-T cell interactions, including antigen presentation, B cell differentiation and antibody production, were profoundly downregulated. Maturation and activation were arrested in a cohort of B cells from pre-stroke RHP-treated mice while regulatory B cells, a subset implicated in neurovascular protection from stroke, were upregulated.
Collectively, our data characterize an endogenous neuroprotective phenotype that utilizes adaptive immune mechanisms pre-stroke to protect the brain from injury post-stroke. Future studies to validate the role of B cells in minimizing injury and promoting central nervous system recovery, and to determine whether B cells mediate an adaptive immunity to systemic hypoxia that protects from subsequent stroke, are needed.

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    ABSTRACT: Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2-week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hrs) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60-min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hrs after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hrs later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hrs to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.
    Preview · Article · Oct 2014 · Journal of Visualized Experiments
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    ABSTRACT: IN PRESS Sterling B. Ortega, Xiagmei Kong, Ramgopal Venkataraman, Allen Michael Savedra, Steven G. Kernie, Ann M. Stowe and Lakshmi Raman Perinatal chronic hypoxia (pCH) is an important risk factor for brain injury and long-term morbidity in children, occurring during the developmental stages of neurogenesis, neuronal migration, and myelination. We show that a rodent model of pCH results in an early decrease in mature myelin. Although pCH does increase progenitor oligodendocytes in the developing brain, BrdU labeling revealed a loss in dividing progenitor oligodendrocytes, indicating a defect in mature cell replacement and myelinogenesis. Mice continued to exhibited hypomyelination, concomitant with long-term impairment of motor function, weeks after cessation of pCH. Implicating a novel neuro-immunological interplay, pCH also induced a significant egress of infiltrating CD4 T-cells into the developing brain. This pCH-mediated neuroinflammation included oligodendrocyte-directed autoimmunity, with an increase in peripheral myelin-specific CD4 T-cells. Thus, both the loss of available mature myelin-producing glial cells, and an active increase in autoreactive, myelin-specific CD4 T-cell infiltration into pCH brains, may contribute to early pCH-induced hypomyelination in the developing CNS. Elucidating potential mechanisms of hypoxia-driven autoimmunity will expand our understanding of the neuro-immune axis during perinatal CNS disease states that may contribute to long-term functional disability.
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    ABSTRACT: IN PRESS Stroke affects millions of people worldwide every year. Despite this prevalence, mechanisms of long-term injury and repair within the ischemic brain are still understudied. Sterile inflammation occurs in the injured brain after stroke, with damaged tissue exposing central nervous system (CNS)-derived antigen that could initiate potential autoimmune responses. We used a standard immunology-based recall response assay for murine immune cells, isolated from the cervical lymph nodes and spleen after transient stroke, to determine if stroke induces autoreactivity to CNS target antigens. Our assays included novel neuronal peptides, in addition to myelin-, nuclear-, glial-, and endothelial-derived peptides. Autoimmune responses to antigen were considered positive based on proliferation and activation over non-stimulated conditions. Stroke induced a significant increase in autoreactive CD4+ and CD8+ T cells, as well as autoreactive CD19+ B cells, as early as 4 days after stroke onset. Mice with large infarct volumes exhibited early T and B cell autoreactivity to NR2A, an NMDA receptor subunit, in cells isolated from lymph nodes but not spleen. Mice with small infarct volumes exhibited high autoreactivity to MAP2, a dendritic cytoskeletal protein, as well as myelin-derived peptides. This autoimmunity was maintained through 10 days post-stroke in both lymph nodes and spleen for all lymphocyte subsets. Sham surgery also induced early autoreactive B cell responses to MAP2 and myelin. Based on these observations, we hypothesize that stroke induces a secondary, complex, and dynamic autoimmune response to neuronal antigens with the potential to potentiate, or perhaps even ameliorate, long-term neuroinflammation.
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