More than 800,000 people in the U.S. will suffer from stroke each year, yet there remains only one FDA- approved acute intervention. Clinical trials to minimize post-stroke inflammation failed, highlighting the need to better understand inflammatory and neuroprotective mechanisms within the central nervous system (CNS). Repetitive hypoxic preconditioning (RHP) naturally protects from stroke for months beyond treatment, minimizing infarct volumes, blood-brain barrier (BBB) disruption, and monocyte, T cell, and neutrophil diapedesis into the ischemic brain. In contrast, RHP specifically induces an immunosuppressed B cell phenotype that is enhanced in the injured CNS of preconditioned mice following stroke. The adoptive transfer of RHP-treated B cells (B(RHP)cells) 6h after transient stroke in male mice reduced infarct volumes and improved functional recovery compared to PBS- or wild type (WT) B cell-treated controls. B(RHP)cell therapy also suppressed a post-stroke autoimmune response to CNS-derived neuronal and myelin antigen (Ag), a novel finding for adaptive autoimmunity that has only been studied in preclinical models of stroke in the context of secondary infection. The goal of this New Investigator grant is to determine if B(RHP)cells, as part of a transferrable adaptive immunity to mild hypoxia, mediate endogenous protection through the production of interleukin 10 (IL10), a cytokine that promotes neuronal repair, as well as interferon (IFN)-?, a cytokine that converts effector (i.e. pro-inflammatory, Th1) T cells to regulatory T cells (Tregs) that suppress other neuropathological immune cells.
Specific Aim 1 will test the hypothesis that B cells confer acute neuroprotection through the production of IL10 and direct neuronal interaction. We will confirm, using state-of-the-art 3-D serial two-photon tomography for whole brain imaging, MRI, and behavioral assays, that IL10- enhanced B(RHP)cells colocalize to a higher number of surviving neurons in the ischemic hemisphere to improve long-term functional recovery. We will also confirm the role of B(RHP)cell-derived IL10 on neuronal viability and axon outgrowth in vitro following oxygen-glucose deprivation (OGD), with B(RHP)cells harvested from IL10-null and WT mice.
Specific Aim 2 will test the hypothesis that B(RHP)cells from WT but not IFN-?-null mice will induce a novel Treg population to suppress pro-inflammatory autoreactive responses from Th1 cells. The transfer of Tregs exposed in vitro to B(RHP)cells will also promote long-term neurovascular and functional recovery (Aim 1A methods), in addition to suppressing CNS-derived autoreactivity. Finally, Glatiramer Acetate, or Copaxone(r), is an FDA-approved drug that upregulates B cell-derived IL10 and Treg conversion in patients with multiple sclerosis.
Specific Aim 3 will test the hypothesis that WT B cells treated with Copaxone will upregulate IL10 and IFN-? to phenocopy B(RHP)cell-mediated protection. We will test the efficacy of Copaxone-treated B cells on neuronal viability in vitro following OGD (Aim 1B methods). Copaxone- or control Betaseron-treated B cells will be adoptively transferred post-stroke to determine efficacy on long-term recovery (Aim 1A methods).

Public Health Relevance

Stroke, the fourth leading cause of death, has few successful clinical treatments to reduce injury due to an incomplete understanding of injury and recovery mechanisms. This proposed research challenges the current dogma that all post-stroke inflammation is detrimental to recovery by identifying a new, immune cell (B cell) population that is a part of the brain's natural protection from stroke (ischemic) injury. These studies will be th first to determine how the transfer of B cells as a post-stroke neurotherapeutic reduces injury and improves recovery, and how this therapy alters a previously unstudied post-stroke autoimmune response that contributes to neuronal protection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS088555-06
Application #
9625666
Study Section
Brain Injury and Neurovascular Pathologies Study Section (BINP)
Program Officer
Bosetti, Francesca
Project Start
2018-12-20
Project End
2020-01-31
Budget Start
2019-02-01
Budget End
2020-01-31
Support Year
6
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Type
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40526
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Selvaraj, Uma Maheswari; Stowe, Ann M (2017) Long-term T cell responses in the brain after an ischemic stroke. Discov Med 24:323-333
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Selvaraj, Uma Maheswari; Poinsatte, Katherine; Torres, Vanessa et al. (2016) Heterogeneity of B Cell Functions in Stroke-Related Risk, Prevention, Injury, and Repair. Neurotherapeutics 13:729-747
Lane-Donovan, Courtney; Desai, Charisma; Pohlkamp, Theresa et al. (2016) Physiologic Reelin does not play a strong role in protection against acute stroke. J Cereb Blood Flow Metab 36:1295-303
Ortega, Sterling B; Noorbhai, Ibrahim; Poinsatte, Katie et al. (2015) Stroke induces a rapid adaptive autoimmune response to novel neuronal antigens. Discov Med 19:381-92