RESEARCH Open Access

Repetitive hypoxic preconditioning induces animmunosuppressed B cell phenotype during

immune mechanisms pre-stroke to protect the brain from injury post-stroke. Future studies to validate the role of B

JOURNAL OF NEUROINFLAMMATION

Monson et al. Journal of Neuroinflammation 2014, 11:22http://www.jneuroinflammation.com/content/11/1/22Southwestern Medical Center at Dallas, 6000 Harry Hines Blvd, Dallas, TX75390-8813, USAcells in minimizing injury and promoting central nervous system recovery, and to determine whether B cells mediatean adaptive immunity to systemic hypoxia that protects from subsequent stroke, are needed.

Keywords: Hypoxic preconditioning, B cells, CXCL13, Stroke, Neuroprotection, B10

* Correspondence: Ann.Stowe@utsouthwestern.eduDepartment of Neurology and Neurotherapeutics, University of TexasConclusions: Collectively, our data characterize an endogendogenous protection from strokeNancy L Monson, Sterling B Ortega, Sara J Ireland, Anouk JM Meeuwissen, Ding Chen, Erik J Plautz, Erin Shubel,Xiangmei Kong, Min K Li, Laura H Freriks and Ann M Stowe*

Abstract

Background: Repetitive hypoxic preconditioning (RHP) creates an anti-inflammatory phenotype that protects fromstroke-induced injury for months after a 2-week treatment. The mechanisms underlying long-term tolerance areunknown, 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 whetherRHP 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) oridentical exposures to 21 % O2 as control. Two weeks following RHP, a 60-minute transient middle cerebral arteryocclusion was induced. Standard techniques quantified CXCL13 mRNA and protein expression. Two days afterstroke, leukocytes were isolated from brain tissue (70:30 discontinuous Percoll gradient) and profiled on a BD-FACSAria flow cytometer. In a separate cohort without stroke, sorted splenic CD19+ B cells were isolated 2 weeks afterRHP and analyzed on an Illumina MouseWG-6 V2 Bead Chip. Final gene pathways were determined using IngenuityPathway Analysis. Students 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 theischemic 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-strokesplenic 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-strokeRHP-treated mice while regulatory B cells, a subset implicated in neurovascular protection from stroke, wereupregulated.

enous neuroprotective phenotype that utilizes adaptive 2014 Monson et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedicationwaiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwisestated.

Monson et al. Journal of Neuroinflammation 2014, 11:22 Page 2 of 18http://www.jneuroinflammation.com/content/11/1/22BackgroundRepetitive hypoxic preconditioning (RHP) is a protocol thatinduces a naturally-protective phenotype in mice prior tostroke onset that defends the central nervous system(CNS) from ischemic damage months beyond the end oftreatment [1]. The benefits of RHP include the attenuationof bloodbrain barrier (BBB) disruption and the reductionof inflammation in the injured, ischemic hemisphere, bothof which contribute to the ultimate extent of strokedamage and infarct volume [2-4]. Given that RHP is asystemic stimulus, it stands to reason that peripheralleukocyte subsets may also be altered in RHP-treatedmice to minimize post-stroke inflammatory responses.In fact, we recently reported that exposing mice to a singlebout of hypoxia, which induces a short-lasting period oftolerance to stroke, significantly increased peripheral Bcell representations within hours in either wild-type ortransgenic mouse strains [5].The role of B cells in stroke is largely overlooked in

preclinical studies, despite the fact that B cell transferinhibits the diapedesis of T cells, neutrophils, monocytes,and macrophages into the ischemic hemisphere, concomi-tant with reduced infarct volumes and functional deficits,in B cell-deficient mice [6-9]. Instead, the common viewremains that B cells do not contribute to the progressionof stroke pathology [10-12], and therefore potentialtherapeutics should focus on the role of T cells in acuteneurovascular injury [10,13,14]. As a result, there arealso no studies investigating the post-stroke role ofCXCL13, a chemokine that specifically recruits B cellsduring inflammation. CXCL13 is upregulated in the CNSby infiltrating immune cells at sites of multiple sclerosislesions [15] and in preclinical studies during CNS infec-tion [16,17]. These data, taken together with the robust Bcell proliferation after one hypoxic exposure, suggest theundiscovered potential for a B cell-mediated protectiveimmunosuppression following RHP treatment in pre-conditioned mice [18].In this study, we tested the hypothesis that RHP creates

a sustained, anti-inflammatory immunophenotype inprotected mice. We investigated the spatial and temporalexpression of CXCL13 message and protein in thepost-stroke brain. We also determined the dynamics ofleukocyte diapedesis into the injured CNS during ischemictolerance to stroke using flow cytometry techniques. Toestablish whether RHP altered adaptive immune mecha-nisms in the healthy animal, we isolated splenic B cellsfrom preconditioned and untreated mice for microarrayanalysis. We found an enhanced representation of B cellsin the post-stroke CNS of RHP-treated mice. We alsoidentified a novel, anti-inflammatory B cell phenotyperesiding for weeks after the last hypoxic exposure an

immunophenotype that may induce protection fromischemic injury in other organ systems, or suppressinflammation during other CNS pathologies. Collectively,these data reveal long-term mechanisms of endogenousCNS protection that include an immunosuppression andreprogramming of B cells occurring prior to stroke onset,with the potential for a novel, B cell-mediated protectionwithin the ischemic cortex of RHP-treated mice.

MethodsRepetitive hypoxic preconditioningThe Institutional Animal Care and Use Committee at UTSouthwestern Medical Center approved all proceduresaccording to the Animal Welfare Act and PHS AnimalWelfare Assurance requirements. Adult, male Swiss Webstermice (Harlan) were randomly assigned to either RHP orcontrol groups at 6 to 7 weeks of age. During RHP, homecages were placed in hypoxic chambers and mice wereexposed to stochastic hypoxic stimuli as previously de-scribed (8% or 11% O2; 2 or 4 hours; nine times over 2weeks) [1], with control animals placed in identicalchambers with 21% O2 (that is, room air) delivered forthe same durations as the RHP group.

Transient middle cerebral artery occlusionAn intraluminal suture was inserted in the common carotidartery to block blood flow to the middle cerebral artery(MCA) in anesthetized animals (2% isoflurane/70% NO2/30% O2) while body temperature was maintained at 37C.Occlusion duration was 60 minutes. Animals were placedinto an incubator (34C) for anesthetic recovery during theocclusion period, then re-anesthetized to withdraw theoccluding suture. Transcranial Laser Doppler flowmetry(Moor Instruments,Wilmington, DE, USA) was used toassess MCA occlusion at the start and end of the occlusionperiod, with occlusion defined as >80% reduction in MCAblood flow relative to baseline for each animal. MCA reper-fusion following suture removal was defined as a return ofblood flow to >50% of baseline. Mice not meeting the bloodflow criteria were removed from the study at the time ofsurgery (17 out of 57 mice, 30%). Sham animals had com-mon carotid artery ligation in the absence of transient mid-dle cerebral artery occlusion (tMCAo). Mice weremonitored for recovery from anesthesia, then returnedto their home cages.

Histology and confocal CXCL13 localization andquantificationAnimals were sacrificed with isoflurane overdose, trans-cardially perfused (20 ml 0.01 M PBS, 40 ml 4% para-formaldehyde), and brains removed for cryoprotection(15% and 30% sucrose). Frozen coronal sections wereprepared, blocked, and stained using standard procedures[1,5]. Primary antibodies and dilutions were: CXCL13

(initial concentration 1:20; final concentration 1:10; R&DSystems, Minneapolis, MN, USA), astrocytes (GFAP 1:200;

Monson et al. Journal of Neuroinflammation 2014, 11:22 Page 3 of 18http://www.jneuroinflammation.com/content/11/1/22Immunostar, Hudson, WI, USA), neurons (NeuN 1:100;Millipore, Billerica, MA, USA) and endothelial cells (CD311:50; BD Pharmingen, San Jose, CA, USA) [5]. Primaryantibodies were detected using Alexa Fluor 488 or 594(1:300; Invitrogen, Grand Island, NY, USA) and nucleiwere counterstained with DAPI. All images were obtainedusing a Leica TCS SP5 confocal laser-scanning microscope(Leica, Buffalo Grove, IL, USA).

Flow cytometrySubmandibular peripheral blood collections were pooledinto heparinized blood collection tubes (3 mice/sample),resuspended in 50 l PBS, and centrifuged at roomtemperature to separate serum from cells. Blood wastransferred to FACS tubes, non-specific binding blockedwith FcRII/III antibody (BD Biosciences, San Jose, CA,USA) and subsequently incubated with the generalleukocyte panel listed below [19] diluted in FACS buffer.Blood was then incubated in FACS Lysing Solution (1:10in dH2O; BD Biosciences) at room temperature for 5minutes, centrifuged (1,500 rpm), and washed again inFACS before analysis on the flow cytometer.Animals were sacrificed with isoflurane overdose and

transcardially perfused with 30 ml 0.01 M PBS as previ-ously described [5] prior to brain dissection. At the time ofperfusion, animals that did not clear all visible blood fromthe contralateral, healthy hemisphere and the sinuses wereexcluded from further flow cytometry analyses (4 out of 31mice, 13 %). Tissue was processed through a 70-m meshscreen, washed in RPMI, and lymphocytes were isolatedwith a 70:30 discontinuous Percoll gradient by centrifuga-tion (2,120 g, 15 minutes, brake off). Lymphocytes wereisolated from brain tissue of either the ischemic orcontralateral cortex, with a pooling of three ischemicor contralateral hemispheres in each sample to ensureadequate leukocyte populations for flow analysis. ThePercoll gradient does not distinguish between resident andinfiltrating immune cells for either condition. After finalRPMI washes, cells were resuspended in 1 ml RPMI onice and cellularity determined by hemocytometer countfor each sample. Nonspecific binding was blocked with anFcRII/III antibody (0.5 l/sample; BD Biosciences). Thegeneral leukocyte panel (BD Biosciences), which does notdistinguish between resident activated microglia and mac-rophages, included CD19 (B cells; 4.0 l/sample), CD11b(monocytes/macrophages; 0.5 l/sample), GR1 (neutro-phils; 0.5 l/sample), CD4 and TCR (T cells; 0.5 l/sample) [19]. For B cell panels, lymphocytes were iso-lated using Lympholyte-M (Cedarlane labs, Burlington,NC, USA), washed with FACS staining buffer, and blockedwith FcRII/III antibody. For the B-cell activation andmaturation panel, cells were labeled with CD19, IgD

(5.0 l/sample), CD93 (2.5 l/sample), CD23 (0.5 l/sample; all eBioscience, San Diego, CA, USA) and CD45R(1.0 l/sample), IgM (2.0 l/sample), and CD138 (10 l/sample; all BD Bioscience) antibodies [20-22]. For theB-regulatory panel, cells were labeled with CD5 (2.0 l/sample; eBioscience), CD45R, CD19, CD1d (0.5 l/sample)antibodies [9,23,24]. After a 45-minute incubation at 4C,cells were washed with FACS wash buffer (PBS with 1%BSA, 0.22 m filtered) and fixed in 1% paraformaldehyde(Electron Microscopy Sciences, Hatfield, PA, USA). Allsamples were run on a BD-FACS Aria flow cytometer usingFACS Diva 6.0 software and data analyzed using FlowJo(Tree Star Inc., Ashland, OR, USA). Hemocytometercounts were used to quantify absolute number.

Quantification of message RNA and protein expressionand microarray analysisBrain lysates were collected at time of flow cytometryanalysis of cortical tissues for quantification of corticalprotein expression. Quantitative immunoassays of theseproteins were performed with standard ELISA kits (R&DSystems) according to the manufacturers instructions. Sep-arate cohorts were transcardially perfused (20 ml RNAse-free PBS), brains removed and flash-frozen for quantitativePCR. CXCL13 mRNA expression was normalized against-actin [1]. In a separate cohort, animals were exposed toRHP or room air and CD19+ B cells were sorted fromspleen at 2 weeks following the last hypoxic exposure. RNAwas isolated and underwent two-rounds of amplificationprior to quantification on an Illumina MouseWG-6 V2array (San Diego, CA, USA) at the UT SouthwesternMicroarray Core. Detectable genes were normalized(Partek Genomics Suite, St. Louis, MO, USA), with finalbetween-group significance of the 18,099 genes detecteddetermined via a restricted maximum likelihood analysisof variance. Gene ontology pathways were analyzed usingIngenuity Pathway Analysis.

Carboxyfluorescein succinimidyl ester-based proliferationAs previously described, but adapted for B-cells, polyclonalresponses were evaluated using the carboxyfluoresceinsuccinimidyl ester (CFSE)-based dilution assay using bulksplenocytes from RHP or control mice [25]. Bulk cellswere suspended at a 1 106/ml concentration in PBSand incubated for 7 minutes with 0.12 M CFSE. Cellswere washed twice with serum-containing media and re-suspended at 1 106/ml concentration in culture media.Cells were activated with lipopolysaccharide (LPS) at 4 g/ml or phorbol myristate acetate at 2 g/ml at 37C in 5%CO2 for 6 days. Subsequently, cells were washed withFACS wash buffer (PBS with 1% BSA, 0.22 m filtered),incubated for 5 minutes at 4C with FcRII/III antibody(BD Bioscience) and labeled with CD19, CD45R, TCRand CD4 fluorescent antibodies. After 45 minutes incuba-

tion at 4C, cells were washed with wash buffer and fixedin 1% paraformaldehyde (Electron Microscopy Sciences).

Flow cytometric data were acquired using a BD-FACSAria flow cytometer using FACS Diva 6.0 software.FlowJo 9.0 software was used to gate on LymphocyteGate+ CD19+CD45+TCR- and polyclonal-specific re-sponses analyzed within the CFSE-low population.

StatisticsAll studies were performed by technicians blinded to ex-perimental condition. All between-group differences wereanalyzed using student t-test or one-way analysis ofvariance with Bonferroni post-hoc analysis (Prism). Signifi-cance was defined as P < 0.05.

ResultsRepetitive hypoxic preconditioning enhances stroke-induced

compared to no-stroke values (P < 0.05). Upregulationof CXCL13 mRNA was not significant in the cortex ofuntreated mice and not observed in the subcortex of eithergroup (Figure 1A). By 2 days following stroke onset, corticalCXCL13 message was elevated in both untreated andRHP-treated groups (both P < 0.01).To determine if RHP affected post-stroke CXCL13

protein expression, we also analyzed cortical lysates col-lected from the ischemic hemispheres to measureCXCL13 protein following stroke induction. One dayafter stroke induction, a >1.5-fold increase in CXCL13protein was found in the ischemic cortex, with the mag-nitude of expression unaffected by prior RHP (bothgroups P < 0.0001 vs sham stroke; Figure 1B). RHP-treated mice maintained a 56 % increase in cortical

tive role for the cerebral endothelium in the expression

13ticaeP).

Monson et al. Journal of Neuroinflammation 2014, 11:22 Page 4 of 18http://www.jneuroinflammation.com/content/11/1/22vascular upregulation of CXCL13 in the ischemic hemisphereThe chemokine CXCL13 specifically recruits B cells fordiapedesis into brain parenchyma, and is upregulated byinflammatory mechanisms in multiple sclerosis [10,15].However, the expression of CXCL13 during CNS inflam-mation within the post-stroke ischemic cortex is unknown.We first sought to determine the baseline mRNA expres-sion of CXCL13, by quantitative real-time PCR, in corticalhomogenates (that is, neocortex and underlying white mat-ter) and subcortical homogenates (for example, striatum,hippocampus, thalamus, and cerebral ventricles). In general,subcortical CXCL13 expression was higher than corticalCXCL13 expression in both untreated (that is, exposed toroom air, or 21% O2 as designated in the Figures) andRHP-treated cohorts (both P < 0.0001; Figure 1A). In theabsence of stroke, RHP completed 2 weeks prior did notsignificantly affect baseline CXCL13 expression in eithercortex or subcortex compared to untreated controls. Oneday after stroke induction, there was a 4-fold upregulationof CXCL13 mRNA in the cortex of RHP-treated mice

Figure 1 CXCL13 is upregulated after stroke. (A) Baseline levels of CXCLhypoxic preconditioning (RHP; n = 12) completed 2 weeks prior in either corhomogenates of the same animals. RHP upregulated cortical CXCL13 messagvalues were elevated in both groups by 2 days (2D; n = 8, 21 % O2; n = 9, RH

cytometry, shows an upregulation at 1 day following stroke (n = 3/group) that rto sham values (n = 5/group). P values and fold change (#X) shown for signifiof the CXCL13 within the ischemic cortex.

Repetitive hypoxic preconditioning maintains B cells inthe ischemic hemisphere with concomitant reductions inother leukocyte subsetsTo determine the impact of RHP on lymphocyte dynamics,we harvested leukocytes to investigate the post-stroke accu-mulation of immune cells in the injured ischemic (that is,site of tMCAo infarction) and uninjured (that is, contra-lateral, contralesional) hemispheres 2 days post-stroke

mRNA (n = 11), prior to transient focal stroke, were unaffected by repetitivel (left of dashed line) or subcortical (right of dashed line) whole brainat 1 day (1D) after stroke onset (n = 6, 21 % O2; n = 7, RHP), though cortical(B) CXCL13 protein, quantified in whole brain lysates collected during flowCXCL13 protein at 2 days following stroke (P < 0.05).Immunohistochemistry revealed a predominantly vascu-lar pattern of CXCL13 protein expression throughoutthe ischemic brain following stroke (Figure 2). Specific-ally, the protein was colocalized largely to CD31+ endo-thelial cells (Figure 2A) within GFAP+ astrocytic endfeetat the BBB (Figure 2B,D), with minimal colocalization toastrocyte cell bodies and non-glial cells, including neu-rons (Figure 2C,E). Together, these data suggest an ac-emains elevated at 2 days only in RHP-treated mice (n = 5/group) comparedcant values. All mRNA and ELISA experiments were run in triplicate.

Figure 2 CXCL13 is predominantly expressed in the cortical endothelium after stroke. Representative confocal images of cortical CXCL13protein (red; 594 nm) colocalized with (A) endothelial cells (CD31; green; 488 nm), (B) astrocytes (GFAP; green) and (C) neurons (NeuN; green) 24hours following stroke at time of peak protein expression. DAPI (blue; 405 nm) was used as a nuclear counterstain and shown in the merge panel.Note the predominately vascular expression of CXCL13 at the bloodbrain barrier in both control mice (top row of each pair) and repetitive hypoxicpreconditioning (RHP)-treated mice (bottom row of each pair). Scale bar = 20 m; n = 3/group; duplicate staining. Zoomed images from adjacentsections show distinct colocalization with (D) CD31 and (E) CXCL13 expression within GFAP + astrocyte endfeet. (F) Zoomed image of the corpuscallosum (pale band within NeuN + staining) shows neurons with punctuate CXCL13 staining denoted by white asterisks. Scale bar for zoomedimages = 30 m. Images were captured using a Leica TCS SP5 confocal microscope with automated DIC optics, a 63 oil objective and LAS AFsoftware, at 21 C. Final figures were created using Adobe Photoshop without gamma adjustments. tMCAo, transient middle cerebral artery occlusion.

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Monson et al. Journal of Neuroinflammation 2014, 11:22 Page 6 of 18http://www.jneuroinflammation.com/content/11/1/22(Figure 3; Additional file 1: Figure S1). Untreated micehad an average of 115,925 leukocytes in the ischemicbrain (Figure 3A), more than double the leukocytespresent in the uninjured hemisphere (50,350 leukocytes;P < 0.05; contralateral scatter plots and all percent distri-bution profiles are shown in Additional file 2: Figure S2).Of the leukocytes that diapedesed into the ischemic cortex,92,399, or 83%, were identified by flow cytometry using ourleukocyte panel, which distinguishes innate (monocytes,macrophages, neutrophils) and adaptive (T helper (CD4+),T cytotoxic (CD8), B cells) immune cells (Figure 3B-G).Leukocyte subsets in the untreated brain following strokewere predominantly monocytes (~48%), B cells (~31%),activated macrophages (~17%), and smaller populations ofneutrophils, CD4+ and CD8 T cells (Figure 3H). In un-treated mice, the contralateral hemisphere leukocyte sub-sets (Figure 3J) consisted predominantly of B cells (~47%),monocytes (~37%), and activated macrophages (~14%).RHP treatment reduced general diapedesis into the

ischemic hemisphere by 41%, to 67,918 total leukocytes(Figure 3A). Populations of CD4+ Tcells (P < 0.01; Figure 3B),monocytes (P < 0.0.05; Figure 3C), and activated macro-phages (P < 0.05; Figure 3D) were lowered by prior RHPto levels indistinguishable from the uninjured hemisphere(Additional file 2: Figure S2B,E,F). However, RHP minim-ally diminished B cell diapedesis (21,730 cells; Figure 3G),that, in conjunction with the decreased diapedesis of othersubsets, increased the percent representation of B cellsfrom 31 % in untreated post-stroke cortex to ~47% of allidentified leukocytes in the protected CNS (Figure 3I).This shift toward increased B cell representation reflectedthe contralateral hemispheres, with only a modestRHP-induced increase from ~47% B cell representationin untreated, uninjured cortex to ~50% in RHP-treated,uninjured cortex (Figure 3K). RHP treatment thereforerestored the ratio of B cells:monocytes (1.3 B cells:monocytes) [26,27] in the ischemic hemisphere comparedto the untreated cohort (0.63 B cells:monocytes; Figure 3L),which was identical to the ratio in contralateral hemi-spheres. Minimal changes in peripheral immunophenotypewere noted (Figure 3M,N; Additional file 3: Figure S3).Taken together, the analysis of percentage and countsrevealed a reversal of cellularity in the ischemic cortex ofRHP-treated mice similar to the pre-clinical conditionsfound in the uninjured cortex.

Repetitive hypoxic preconditioning induces animmunosuppressed phenotype in resident B cells prior tostroke onsetTo assess if RHP could alter B cell immunophenotypeprior to stroke onset, resident splenic B cells were isolatedfor microarray analysis 2 weeks following the completion

of RHP (Additional file 4: Figure S4). We discovered thatthe CD19+ B cells exhibited gene expression profiles witha distinct clustering (Figure 4A) from four out of fiveRHP-treated mice (excluding RHP #5). B cells from RHP-treated mice had altered expression of over 1,900 genescompared to 18,099 genes identified in untreated controlB cells, with the top 10 up- and downregulated geneslisted in Figure 4B (top 50 up- and downregulated geneslisted in Tables 1 and 2). Figure 4C shows the canonicalpathways activated in RHP-treated B cells, which includea predominant downregulation of genes expressed by Bcells that are important for T cell differentiation and sig-naling. Interestingly, seven of the top canonical pathwaysinclude key components in B-T cell interactions thatwould significantly alter inflammatory responses. Reana-lyzing the data set to focus on B-T cell interactionshighlighted significant downregulation (all z scores > 2)of the quantity of T cells (P = 7.4E-05), differentiation ofCD4 T cells (P = 2.6E-04), and the immune response ofcytotoxic CD8 T cells (P = 2.1E-03). Both CD4 and CD8 Tcell subtypes have been previously implicated in the pro-gression of neurovascular injury following stroke [4,10,14]and remain downregulated in ischemic hemisphere ofRHP-treated mice after stroke (Figure 3). In addition,upstream analysis showed a predicted activation ofinterleukin-4 (P = 1.5E-02; Figure 5A) and subsequentinhibition of the downstream transcriptional regulatorsinterferon regulatory factor (IRF)-1, IRF4, IRF8, and thecytokine TNF-.The RHP-induced regulation of CD4 T cell differentiation

(21/68 genes differentially affected; 52% of altered genesdownregulated; P = 3.0E-06) included a staggering 6,800-fold downregulation of histocompatibility 2, O regionbeta locus (H2-Ob), the homolog to human HLA-DOB(Figure 5B). HLA-DOB is selectively expressed in B cellsand required for B cell antigen presentation under thetranscriptional regulation of the major histocompatibilitycomplex (MHC) class II transactivator CIITA [28], with themouse homolog Ciita also downregulated in RHP-treated Bcells (1.68-fold; P = 4.6E-06). The canonical pathwaywith the greatest downregulation and second-highestsignificance was the antigen presentation pathway (13/29genes differentially affected; 85% of altered genes down-regulated; P = 3.5E-06). Other B cell-specific biologicalfunctions significantly downregulated (all z-scores > 2)include hematopoiesis, development and differentiationof lymphocytes, and the production of antibody (Figure 5C).These microarray data suggest that RHP suppresses theproliferation, development, and differentiation of B cells,in addition to inhibiting B-T cell adaptive immune interac-tions, weeks after completion of RHP and prior to strokeonset.To gain a deeper understanding of our microarray

results, we characterized splenic B cell populations from

RHP-treated mice and performed an ex vivo phenotypeanalysis using flow cytometry. As B cells mature, they

Figure 3 (See legend on next page.)

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(See figure on previous page.)Figure 3 Repetitive hypoxic preconditioning recruits B cells into the ischemic cortex following stroke. (A-G) Overall leukocyte diapedesiswas diminished in the ischemic cortex of repetitive hypoxic preconditioning (RHP)-treated mice (blue circles; n = 15) compared to control miceexposed to room air (21 % oxygen) without preconditioning (black circles; n = 12). Specifically, RHP reduced the mean populations (blue bars) ofCD4+ T cells, monocytes, and macrophages in the ischemic hemispheres to levels indistinguishable from the contralateral hemispheres (shown inAdditional file 2: Figure S2). Mean shown below each graph in thousands. (H-N) The distribution of identified leukocyte subsets from the (H,I)ischemic cortex and (J,K) contralateral (that is, uninjured) cortex (Additional file 2: Figure S2), with (L) ratios identified. (M,N) Peripheral blood ofthe same animals (Additional file 3: Figure S3) is shown, with total number of immune cells shown below. RHP-treated mice clearly have anincreased recruitment of B cells in the ischemic cortex. Mean SD. Data represent four independent experiments.

Figure 4 Repetitive hypoxic preconditioning induces an immunosuppressive phenotype prior to stroke. B cells isolated from spleen wereanalyzed with microarray, showing (A) distinct clustering of repetitive hypoxic preconditioning (RHP)-treated mice compared to controls (21 %;n = 5/group). (B) Top 10 up- and downregulated genes, and (C) top activated canonical pathways are shown. Gene products are shown in bargraph form as percentages of all genes associated with a listed pathway (upper axis). Gene numbers up/downregulated are located in the bars, withthe total number of genes within the pathway shown to the right. Expression changes are shown as either upregulated (red bars) or downregulated(green bars), and pathways with B-T cell interactions are marked by a cross. Respective P values are indicated by orange squares (lower axis). Replicatesamples on the microarray chip are indicted with (R) in panel A.

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Table 1 Top 50 upregulated genes isolated from repetitive hypoxic preconditioning-treated splenic B cells comparedto untreated splenic B cells

Accession Symbol Fold change P value Abbreviated definition

NM_010474.1 Hs3st1 14.00 1.38E-09 Heparan sulfate (glucosamine) 3-O-sulfotransferase 1 (Hs3st1)

NM_013498 Crem 7.90 4.02E-02 cAMP responsive element modulator (Crem), transcript variant 3

NM_013498 Crem 7.74 1.93E-02 cAMP responsive element modulator (Crem), transcript variant 3

NM_145552 Gnl2 4.70 2.78E-02 Guanine nucleotide binding protein-like 2 (nucleolar) (Gnl2)

NM_026301.1 Rnf125 4.61 4.29E-02 Ring finger protein 125 (Rnf125)

NM_027950.1 1700012B18Rik 4.35 1.76E-08 Oxidative stress induced growth inhibitor 1 (Osgin1), nuclear geneencoding mitochondrial protein

NM_013498.1 Crem 4.21 1.57E-02 cAMP responsive element modulator (Crem), transcript variant 3

AK053379 E130012P04Rik 4.13 1.34E-02 CDNA FLJ20330 FIS, CLONE HEP10296 homolog [Homo sapiens],full insert sequence

NM_009368.1 Tgfb3 4.07 2.58E-02 Transforming growth factor, beta 3 (Tgfb3)

XM_144142.3 1810045K06Rik 3.72 5.20E-04 Kelch-like 21 (Klhl21)

NM_019980 Litaf 3.58 1.05E-02 LPS-induced TN factor (Litaf)

NM_010831.1 Snf1lk 3.57 2.21E-04 Salt inducible kinase 1 (Sik1)

XM_138460.2 LOC218060 3.23 4.61E-02 -This record was discontinued.

NM_177091.2 Fndc7 3.16 3.81E-04 Fibronectin type III domain containing 7 (Fndc7), transcript variant 1

NM_177876.2 BC026744 3.16 2.07E-07 Vacuolar protein sorting 37B (yeast) (Vps37b)

NM_145222.1 B3gnt7 3.12 8.64E-11 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase7 (B3gnt7)

NM_178149.2 1500004A08Rik 3.04 1.76E-06 Phosphoinositide-3-kinase interacting protein 1 (Pik3ip1)

NM_053272 Dhcr24 2.92 4.08E-03 24-dehydrocholesterol reductase (Dhcr24)

XM_354933.1 2410015A16Rik 2.87 2.41E-02 Lymphocyte antigen 6 complex, locus K (Ly6k)

NM_145559.1 Slc2a9 2.87 1.70E-02 Solute carrier family 2 (facilitated glucose transporter), member 9(Slc2a9), transcript variant 4

AK037598 Stat4 2.82 4.05E-02 Signal transducer and activator of transcription 4, full insert sequence

NM_007393.1 Actb 2.77 1.08E-10 Actin, beta (Actb)

NM_134109.1 Ildr1 2.76 6.72E-03 Immunoglobulin-like domain containing receptor 1 (Ildr1)

NM_011742.1 Zfp1 2.75 5.42E-03 Zinc finger protein 1 (Zfp1), transcript variant 2

AK047415 B930059G07Rik 2.75 7.90E-03 HEMATOPOIETIC CELL PROTEIN-TYROSINE PHOSPHATASE70Z-PEP (EC 3.1.3.48)

NM_009883.1 Cebpb 2.73 3.37E-05 CCAAT/enhancer binding protein (C/EBP), beta (Cebpb)

NM_007393.1 Actb 2.65 4.10E-10 Actin, beta (Actb)

AK038223 A130087I02Rik 2.65 1.16E-02 RIKEN full-length enriched library, clone:A130087I02 product:unclassifiable, full insert sequence

XM_148086.4 LOC224137 2.64 8.14E-03 -This record was discontinued.

AK053596 E130112N23Rik 2.63 4.30E-05 RIKEN full-length enriched library, clone:E130112N23 product:hypothetical protein, full insert sequence

NM_026301.1 Rnf125 2.63 4.68E-02 Ring finger protein 125 (Rnf125)

NM_027013.1 Scnm1 2.61 1.07E-02 Sodium channel modifier 1 (Scnm1), transcript variant 1

NM_144554.1 Trib3 2.55 7.58E-03 - tribbles homolog 3 (Drosophila), validated

AK039925 A430031F07Rik 2.54 3.29E-03 RIKEN full-length enriched library, clone:A430031F07 product:hypothetical protein, full insert sequence

XM_358387.1 Crsp2 2.53 2.36E-02 Mediator complex subunit 14 (Med14), transcript variant 2

NM_172934.1 4632417D23 2.47 4.75E-04 -"This RefSeq was permanently suppressed because currentlythere is insufficient support for the protein."-

AK041320 Icosl 2.47 4.97E-05 Icos ligand, full insert sequence

Monson et al. Journal of Neuroinflammation 2014, 11:22 Page 9 of 18http://www.jneuroinflammation.com/content/11/1/22

hy

3

3

2

4

8

5

7

2

2

4

3

1

3

Monson et al. Journal of Neuroinflammation 2014, 11:22 Page 10 of 18http://www.jneuroinflammation.com/content/11/1/22progressively increase their expression of MHC class IIand thus increase their ability to interact with T cells[22]. We therefore evaluated the ex vivo maturationstatus of splenic B cells by first evaluating the frequency oftransitional (T1, T2 and T3) B cells. T1 B cells do not mi-grate to lymph nodes and, while T3 B cells express higher

Table 1 Top 50 upregulated genes isolated from repetitiveto untreated splenic B cells (Continued)

NM_009546.1 Trim25 2.47 2.45E-0

NM_180974.1 Foxn2 2.46 1.09E-0

NM_172442.2 Dtx4 2.42 2.23E-0

NM_019518.2 Grasp 2.41 1.48E-0

AK052205 D330008I21Rik 2.39 3.03E-0

NM_134109.1 Ildr1 2.38 1.33E-0

NR_027901.1 2900060B14Rik 2.37 3.51E-1

2010204O13Rik 2.36 4.00E-0

NM_025549.1 Arrdc4 2.35 4.64E-0

AK041549 A630020I15Rik 2.32 6.38E-0

NM_029083.1 Ddit4 2.32 3.87E-0

AK020047 6030405P19Rik 2.29 2.67E-1

NM_009843.2 Ctla4 2.27 4.44E-0levels of B220, they are distinct from mature B cells [22].Gating on CD19+CD93+ B cells and using IgM versusCD23 in order to discriminate between the transitionalpopulations (Additional file 5: Figure S5), we observed asignificant increase in T1 cells isolated from RHP-treatedmice compared to untreated mice (14.32% vs 11.70%,respectively; P < 0.01; Figure 6A). RHP therefore reducedthe resident pool of B cells capable of antigen presenta-tion, as MHC class II and CD21/CD35 expression islowest in the T1 stage [22]. RHP also inhibits fully acti-vated B cell status by inducing a concomitant increase inearly (IgM+IgD-CD19+) and mid-phase (IgM+IgD+CD19+)B cells (3.24% vs 5.3%; P < 0.01, 14.52% vs 22.07%; P 2. All significance and graphs generated by Ingenuity Pathway Analysis software.

Monson et al. Journal of Neuroinflammation 2014, 11:22 Page 14 of 18http://www.jneuroinflammation.com/content/11/1/22Figure 6 Repetitive hypoxic preconditioning treatment modulates thesystem injury. (A) Transitional state of CD19+CD93+ B-cells were quantifiecells. Repetitive hypoxic preconditioning (RHP) suppresses the maturation owas analyzed by quantifying the level of early (IgM+IgD-), mid (IgM+IgD+) oin the resident B-cells. (C) Conventional B-cell subtypes such as marginal zoCD93- populations and not affected by RHP. (A-C) n = 6/group; two indepeassessed using carboxyfluorescein succinimidyl ester (CFSE) dilution assay w(dPF) is the percentage of CFSE low cells in the test condition (stimulated) mitwo independent experiments with n = 4 per condition. Mean percentages peripheral B-cell immune compartment prior to central nervousd and measured as T1 (IgM+CD23-), T2 (IgM+CD23+) or T3 (IgM-CD23+)f B-cells from T2 to T3 stage. (B) Ex vivo splenic B-cell activation statusr late (IgM-IgD+) CD19+ B-cells. RHP inhibits fully activated B-cell statusne (MZ) and follicular B-cells (FOB) were quantified within the CD19+

ndent experiments. (D) In vitro polyclonal B cell responses wereith lipopolysaccharide (LPS) stimulation. Delta proliferation fractionnus the background (non-stimulated condition). Data is representative ofSD are shown. 21 %, Untreated cohorts; PMA, phorbol myristate acetate.

inepe2 (in

Monson et al. Journal of Neuroinflammation 2014, 11:22 Page 15 of 18http://www.jneuroinflammation.com/content/11/1/22receptor, in patients at 1 day after stroke is highly predict-ive of post-stroke infection, which impeded long-term re-covery of these patients [45] and emphasizes thecomplexity of endogenous adaptive immunity followingRHP.Upon exiting the bone marrow, B cells are specifically

recruited to the spleen where they mature to proliferate,produce antibodies and secrete cytokines when activatedby antigen. When antigen is removed, a selected fewremain in a sustained memory state until a rapid responseis needed upon subsequent presentation of the antigenduring adaptive immunity [46]. Antibody production istypically studied under states of infection or auto-immunedisorders, but little is known about antibody-producingplasma cell immune responses during cerebral ischemiaor endogenous neurovascular protection. In our model,the immune function with the greatest RHP-inducedmodulation prior to stroke was the downregulation of

Figure 7 Repetitive hypoxic preconditioning treatment specificallyto central nervous system injury. Ex vivo regulatory B-cell levels from r(21 %) cohorts. B10 (CD1dhiCD5+), B1a (CD1d+CD5+) and conventional BB-cells populations. Mean percentages SD are shown; n = 6/group; twothe antibody response. In addition, the transcriptionalrepressor B cell lymphoma (Bcl)-6 and Bcl-6 interact-ing co-repressor are both significantly downregulated(Table 2), possibly to minimize germinal center develop-ment and thus antibody-mediated immune responses [46]should injury occur. RHP decreased the repertoire ofmature B cells, while increasing regulatory B cells, byincreasing T1 and IgM+IgD- B cells, with concomitantreduction of IgM-IgD+ B cells, an effect which may beoccurring through either inhibition of B cell maturationand/or selective depletion of mature cells. Reduction inperipheral mature B cells (that is, IgD-expressing cells)induces tolerance in murine autoimmune studies andpan-mature B-cell depletion in human CNS diseases[47-49]. Although transitional B cells are capable of anti-gen presentation, they were found to be poor activators ofnave CD4 T-cells [50].We hypothesize that RHP inhibits B cell-mediated

immune mechanisms, which potentially promote acuteinjury, while simultaneously augmenting disease-inhibitingregulatory B cells, a complex interplay of adaptive immunemechanisms also identified in autoimmune disease [51]that will be addressed in future studies. It remains un-clear whether antibodies are produced during the initialexposures to hypoxia at the start of RHP to mediateadaptive immunity resulting in subsequent post-strokeimmunosuppression [18]. Furthermore, RHP is systemicstimuli that may influence B cell development in thebone marrow compartment, which serves as a contem-poraneous B cell maturation and activation site to thespleen [52]. The egress of mature B cells from the bonemarrow typically occurs within 5 days, a more rapid ratethan during splenic maturation [52]. Therefore, our ana-lysis of stable, immature B cell phenotypes at 14 days afterRHP in the spleen most likely reflects a similar profilewithin the bone marrow, though this observation needsto be experimentally confirmed. Future studies will also

duces an immunosuppressive regulatory B-cell population priortitive hypoxic preconditioning (RHP)-treated mice relative to untreatedCD1dlowCD5-) subpopulations were quantified within splenic CD19+

dependent experiments.elucidate the role of antigen presentation and antibodyproduction in the establishment of endogenous neuro-vascular protection, in addition to the progression ofpost-stroke injury and repair.Our findings identified the upregulation of CXCL13

within the BBB in response to stroke (Figures 1 and 2).Stroke induced a predominantly vascular expression ofCXCL13 protein throughout the ischemic hemisphere,though others show both astrocytes [53] and stimulatedneurons [54] can upregulate this chemokine during in-flammation. While early upregulation of CXCL13 mRNAin RHP-treated mice could potentially allow for theselective recruitment of post-stroke B cells into the pro-tected brain in the absence of other leukocyte populations,CXCL13 mRNA is equally upregulated in the untreatedischemic BBB by 2 days following stroke onset. Thismay prove beneficial in future studies investigating thepotential for the transfer of B cell-mediated neurovascu-lar protection to nave mice after stroke, as selective

diapedesis. (A-G) Percent representation (top panel) and total count (bot-

representation in peripheral blood and spleen was quantified and

significant in (C) splenic leukocyte counts. While RHP-treated mice had

independent experiments.

of Texas Southwestern Medical Center at Dallas).

Monson et al. Journal of Neuroinflammation 2014, 11:22 Page 16 of 18http://www.jneuroinflammation.com/content/11/1/22recruitment of B cells from the periphery will not be asole by-product of the RHP treatment. It will also improvethe translation of pro-recovery B cells as a potentialneurotherapeutic if we can determine if the presence ofreprogrammed B cells in the ischemic cortex promoteslong-term recovery mechanisms, such as neuronal plasticityand angiogenesis. Activated, but not resting, B cells arethe major producers of lymphocyte-derived brain-derivedneurotrophic factor in mouse and humans [54,55] and, asbrain-derived neurotrophic factor secretion by B cellssupports neuronal survival both in vitro [54] and in vivo[56,57], it is imperative to discover in future studies ifRHP alters post-stroke B cells to enhance the upregulationof this, and other growth factors, in the post-stroke brain.

ConclusionsWith this data, we describe for the first time the expressionpattern of the chemokine CXCL13 following stroke, andthe novel maintenance of B cells in the protected ischemiccortex. We also identified an adaptive B cell immuno-phenotype following brief systemic hypoxia that includesdownregulation of antigen presentation and antibody pro-duction within the quiescent immune system. In humans,living at high altitudes [58], pre-stroke exercise [59], andprior limb ischemia [60-62] all mitigate the severity ofischemic injury. It is unknown how these systemic precon-ditioning interventions reduce injury, though a recent clin-ical study shows that remote ischemia confers myocardialprotection in infants through the upregulation of IL-10[62]. Our observation of RHP-induced B cell recruitmentmay be the first step to understanding the role of adaptiveimmunity in mitigating the severity of ischemia or otherinjuries within the CNS and other organs. Ultimately,understanding the mechanisms by which the CNS choosesto protect itself is foundational to developing effectivetherapies, not only for the treatment of stroke-relatedinjury, but potentially for other diseases that involve eithera loss of BBB integrity or the recruitment of leukocytesinto the parenchyma.

Additional files

Additional file 1: Figure S1. Gating strategies for the ischemic cortexand peripheral blood samples following stroke. Gating strategies from (A)perfused ischemic and contralateral hemispheres and (B) peripheralblood. Specific leukocyte populations are identified in the gating.

Additional file 2: Figure S2. Repetitive hypoxic preconditioning (RHP)continues to block leukocyte diapedesis into the ischemic cortex at 2days following stroke. (A-G) Percent representation (top panel) and totalcount (bottom panel) shown for each gated leukocyte subset for theischemic (filled circles) and contralateral (open circles) cortex. RHP decreasedleukocyte diapedesis into the ischemic cortex of RHP-treated mice (bluesymbols) at 2 days after transient middle cerebral artery occlusion (tMCAo)compared to control mice without preconditioning (21%; black symbols;

n = 3 animals/point; n = 1221 animals/group; hemocytometer counts werenot collected for one experiment). RHP reduced CD4+ T-cells, monocytes,AbbreviationsBBB: bloodbrain barrier; Bcl: B cell lymphoma; BSA: bovine serum albumin;CFSE: carboxyfluorescein succinimidyl ester; CNS: central nervous system;DAPI: 4',6-diamidino-2-phenylindole; dPF: delta proliferation fraction;ELISA: enzyme-linked immunosorbent assay; Ig: immunoglobulin;IL: interleukin; IRF: interferon regulatory factor; LPS: lipopolysaccharide;MCA: middle cerebral artery; MHC: major histocompatibility complex;PBS: phosphate-buffered saline; PCR: polymerase chain reaction;RHP: repetitive hypoxic preconditioning; tMCAo: transient middle cerebralartery occlusion; TNF: tumor necrosis factor.

Competing interestsThe authors declare that they have no competing interests.

Authors contributionsNLM, SJI, and AMS designed the original CNS flow cytometry experiments,which were conducted by NLM, SJI, MKL, LHF, and AMS. The B cell microarraywas analyzed by AMS. B cell-specific flow cytometry panels were designed andexecuted by SBO, AJMM, DC, and XK. EJP and AMS conducted all tMCAoprocedures, and ES conducted quantitative PCR. XK and MKL conductedprotein assays and histology. SBO, SJI, DC, and AMS were responsible fordata analysis. All authors read and approved the final manuscript.

AcknowledgmentsThe authors would like to thank Charlene Supnet for help with editing thismanuscript, and Julia Hankins for help in early B cell panel development. Thesestudies would also not be possible without the Neuro-Models Facility, Departmentof Neurology & Neurotherapeutics, University of Texas Southwestern MedicalCenter at Dallas, and The Haggerty Center for Brain Injury and Repair (Universitymore splenic leukocytes, (D) overall distributions of subsets were similar.B cells isolated for microarray analysis are designated by the red outline.*P < 0.05 vs 21% O2 controls. Data were collected on the same day formicroarray analysis.

Additional file 5: Figure S5. Flow cytometric analysis of B-cellimmunophenotype after repetitive hypoxic preconditioning (RHP) modulation.(A) Gating strategies for assessment of B-cell maturation and activation inRHP-treated resident B-cells (n = 6) relative to untreated control (21%)cohorts (n = 6). (B) Gating strategy to quantify regulatory (B10 and B1a)and conventional (B2) B-cells in RHP treated mice. Data represent twocompared to 21% O2 controls (black symbols; n = 5). RHP diminishedpercent representation of B cells in blood, and CD8 T cells in spleen. Atrend for an increase in neutrophil representation in the spleen wastom panel) shown for each gated leukocyte subset quantified inperipheral blood. Only (F) neutrophil and (G) B-cell representations wereaffected by prior RHP (n = 12) compared to control (n = 24). Distributionof leukocyte subsets for cellular leukocyte counts are shown in Figure 3.Mean (bars) standard deviation (SD; whiskers); *P < 0.05 vs untreatedmice.

Additional file 4: Figure S4. Repetitive hypoxic preconditioning (RHP)minimally alters peripheral lymphocyte representation prior to stroke.Two weeks following RHP (blue symbols; n = 5), (A,B) leukocyteand macrophages in the ischemic hemispheres to levels indistinguishablefrom the contralateral hemispheres. Mean (bars) standard deviation (SD;whiskers); *P < 0.05; **P < 0.01; *vs contralateral cortex unless otherwisedesignated by a horizontal bar.

Additional file 3: Figure S3. Repetitive hypoxic preconditioning (RHP)recruits B-cells from the periphery while actively blocking neutrophilReceived: 25 October 2013 Accepted: 13 January 2014Published: 31 January 2014

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doi:10.1186/1742-2094-11-22Cite this article as: Monson et al.: Repetitive hypoxic preconditioninginduces an immunosuppressed B cell phenotype during endogenousprotection from stroke. Journal of Neuroinflammation 2014 11:22.Submit your manuscript at www.biomedcentral.com/submit

AbstractBackgroundMethodsResultsConclusions

BackgroundMethodsRepetitive hypoxic preconditioningTransient middle cerebral artery occlusionHistology and confocal CXCL13 localization and quantificationFlow cytometryQuantification of message RNA and protein expression and microarray analysisCarboxyfluorescein succinimidyl ester-based proliferationStatistics

ResultsRepetitive hypoxic preconditioning enhances stroke-induced vascular upregulation of CXCL13 in the ischemic hemisphereRepetitive hypoxic preconditioning maintains B cells in the ischemic hemisphere with concomitant reductions in other leukocyte subsetsRepetitive hypoxic preconditioning induces an immunosuppressed phenotype in resident B cells prior to stroke onsetRepetitive hypoxic preconditioning induces a regulatory B-cell population

DiscussionConclusionsAdditional filesAbbreviationsCompeting interestsAuthors contributionsAcknowledgmentsReferences

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