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


METHODS
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).


RESULTS
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.


CONCLUSIONS
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.
Nancy L
NL
Monson
Sterling B
SB
Ortega
Sara J
SJ
Ireland
Anouk Jm
AJ
Meeuwissen
Ding
D
Chen
Erik J
EJ
Plautz
Erin
E
Shubel
Xiangmei
X
Kong
Min K
MK
Li
Laura H
LH
Freriks
Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Blvd, Dallas, TX 75390-8813, USA. Ann.Stowe@utsouthwestern.edu.
Ann M
AM
Stowe
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism* / Q000473:pathology; D049109:Cell Proliferation; D054360:Chemokine CXCL1 / Q000378:metabolism; D004195:Disease Models, Animal; D004727:Endothelium / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D007839:Functional Laterality; D005786:Gene Expression Regulation / Q000502:physiology; D007165:Immunosuppression Therapy; D020244:Infarction, Middle Cerebral Artery / Q000150:complications*; D019194:Ischemic Preconditioning; D008297:Male; D051379:Mice; D046228:Microarray Analysis; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D013997:Time Factors
D015703:Antigens, CD; D054360:Chemokine CXCL1; D010751:Phosphopyruvate Hydratase