ARTICLE OPEN ACCESS

Harmful neutrophil subsets in patients withischemic strokeAssociation with disease severity

David Weisenburger-Lile MD Yuan Dong PhD Marion Yger MD Gaelle Weisenburger MD

Giulia Frasca Polara MD Thomas Chaigneau MS Riccardo Zapata Ochoa MS Beatrice Marro MD

Bertrand Lapergue MD PhD Sonia Alamowitch MD and Carole Elbim MD PhD

Neurol Neuroimmunol Neuroinflamm 20196e571 doi101212NXI0000000000000571

Correspondence

Dr Carole Elbim

caroleelbimupmcfr

AbstractObjectiveTo better understand the functional state of circulating neutrophils in patients with ischemicstroke (IS) for planning future clinical trials

MethodsWe analyzed by flow cytometry activation state of circulating neutrophils and the distribution ofneutrophil peripheral subsets in 41 patients with acute IS less than 6 hours before admission andcompared them with 22 age-matched healthy controls

ResultsOur results demonstrated continuous basal hyperactivation of circulating neutrophils duringacute IS characterized by lower L-selectin expression and higher CD11b expression at the cellsurface increased ROS production by neutrophils and greater circulating levels of neutrophilelastase Neutrophil hyperactivation was associated with deregulation of the equilibrium be-tween apoptotic and necrotic Patients also had higher percentages than controls of theoveractive senescent (CXCR4brightCD62Ldim) neutrophil subset and increased percentage ofneutrophils with a reverse transendothelial migration (CD54highCXCR1low) phenotype Im-portantly neutrophil alterations were associated with the clinical severity of the stroke eval-uated by its NIH Stroke Scale score

ConclusionAltogether our results indicate that during acute IS the inflammatory properties of circulatingneutrophils rise associated with the expansion of harmful neutrophil subsets These changes inneutrophil homeostasis associated with disease severity may play an instrumental role bycontributing to systemic inflammation and to the blood-brain barrier breakdown Our findingshighlight new potential therapeutic approaches of stroke by rebalancing the ratio of senescentto immunosuppressive neutrophils or decreasing reverse neutrophil transmigration or both

RELATED ARTICLE

EditorialContribution ofpolymorphonuclearneutrophils in the bloodperiphery to ischemic braininjury

Page e570

From the Sorbonne Universites (DW-L YD TC RZO SA CE) UPMC Univ Paris 06 UMRS 938 CdR Saint-Antoine Hopital Saint-Antoine INSERM (DW-L YD TC RZO SACE) UMRS 938 CdR Saint- Antoine Team ldquoImmune System Neuroinflammation and Neurodegenerative Diseasesrdquo Hopital St-Antoine Service de Neurologie et drsquoUrgencesNeurovasculaires (DW-L MY SA) Assistance Publique-Hopitaux de Paris Hopital Saint-Antoine Division of Pneumology (GW) Foch Hospital F-92150 Suresnes Division ofNeurology (GFP) Stroke Center Assistance Publique-Hopitaux de Paris Pitie-Salpetriere Hospital Division of Radiology (BM) Assistance Publique-Hopitaux de Paris Hopital Saint-Antoine and Division of Neurology (BL) Stroke Center Foch Hospital F-92150 Suresnes

Go to NeurologyorgNN for full disclosures Funding information are provided at the end of the article

The Article Processing Charge was funded by the authors

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Cerebral ischemia elicits a strong inflammatory response in-volving the transmigration of several subsets of leukocytesincluding neutrophils1 Murine models of ischemic stroke(IS) reveal that neutrophils are the first cells recruited into thebrain minutes after stroke accompanied by local productionof cytokines or chemokines and facilitated by a very earlyischemia-induced breakdown in the blood-brain barrier(BBB)23 Massive infiltration of neutrophils into ischemicbrain tissue has also been documented in human strokepatients4 Although animal stroke models suggest that de-pleting circulating neutrophils before or at the onset of strokereduces infarct size human drug trials aimed at preventingpotentially harmful subacute neutrophil adhesion and in-filtration into the brain parenchyma poststroke have not im-proved patient recovery56 The failure of these trials might bepartially explained by the undiscriminating nature of theseapproaches the anti-inflammatory potential of some circulatingneutrophil subsets means that globally blocking neutrophilaccess to the brain might not be a viable therapeutic approachA better understanding of the functional state of circulatingneutrophils in patients with IS is therefore crucial for planningfuture clinical trials targeting postischemic inflammation

Neutrophils the most abundant circulating leukocytes playa key role in the immune defense against bacterial and fungalpathogens They are rapidly recruited from blood to sitesof infection where they kill microorganisms via a number ofdifferent mechanisms including phagocytosis production ofreactive oxygen species (ROS) release of granular proteinsneutrophil extracellular traps (NETs) a process calledNETosis However in case of inappropriate andor excessiveactivation neutrophils play also a key role in the induction orpromotion of inflammation-induced tissue injury7 Recentstudies point to the existence of heterogenous populations ofmature and immature neutrophils In fact diverse subsets ofneutrophils differ markedly in their proinflammatory activitySenescent neutrophils an overly active subset express ele-vated levels of CXCR48 whereas the immunosuppressiveCD16brightCD62Ldim subset exhibits reduced proin-flammatory properties9 Recent studies also demonstrate thatneutrophils can migrate back into the circulation in a processreferred to as reverse transendothelial migration (rTEM)10

Contradictory results on the phenotype and functions of circu-lating neutrophils in patients with IS have been reported Someauthors report fewer oxidative bursts and NET production11

whereas others suggest increased degranulation and ROS pro-duction12 Some analyzed neutrophil functions 24ndash48 hoursafter IS although it is now clear that inflammatory pathways areinvolved in the first hours1 None has investigated the distribu-tion of the senescent immunosuppressive and rTEMneutrophilsubsets or their consequences on the course of IS

Our study performed in whole blood to avoid artifacts due toisolation procedures13 aimed to characterize fully the phe-notypes and functions of human peripheral neutrophils atdifferent stages of acute IS We analyzed neutrophil activationstate and the distribution of 3 neutrophil subsets the sen-escent subset the immunosuppressive subset and the rTEMsubset in the aim of addressing the right targets for planningfuture clinical trials We investigated potential correlationsbetween neutrophil parameters and clinical markers of diseaseprogression

MethodsStudy designThis study enrolled 41 patients with acute IS hospitalized inthe stroke unit of Saint-Antoine Hospital Pitie-SalpetriereHospital (both Paris France) and Foch Hospital (SuresnesFrance) during the acute phase of brain ischemia Patientswere included if they had had a stroke less than 6 hours beforeadmission regardless of the NIH Stroke Scale (NIHSS) scorewere aged ge18 years and showed no signs of infection at theadmission All patients were examined by neurologists ad-mitted to the stroke unit and treated according to acceptedstroke guidelines14 We prospectively collected the followingdata for each with a structured questionnaire vascular riskfactors (age sex presence of atrial fibrillation hypertensiondiabetes hypercholesterolemia previous stroke history andtreatment) stroke severity assessed by the NIHSS at admis-sion (day 0) day 2 and day 7 and time from symptom onsetto brain imaging and sampling The modified Ranking scale(mRs) was used for functional disabilitydependence evalu-ations at 3 months Standard stroke evaluation included brainimaging (MRI performed for 37 patients andCT performed for4 patients when MRI was contraindicated) blood sampling(especially neutrophil and lymphocyte counts) electrocardi-ography for at least 48 hours transthoracic echocardiographyand vessel imaging by ultrasound MR angiography or angio-CT We prospectively collected stroke information includinglocation of ischemia in the brain presence and location of

GlossaryAAD = amino-actinomycin D ANOVA = analysis of variance APC = allophycocyanine BBB = blood-brain barrier DAMP =danger-associated molecular pattern fMLP = N-formylmethionyl-leucyl-phenylalanine HC = healthy control HE =hydroethidine HMGB = high-mobility group box IS = ischemic stroke LPS = lipopolysaccharide MMP = matrixmetalloproteinaseMPO =myeloperoxidaseNET = neutrophil extracellular trapNIHSS =NIH Stroke ScaleNLR =Nod-likereceptor PBS = phosphate buffered saline PMN = polymorphonuclear neutrophil ROS = reactive oxygen species rTEM =reverse transendothelial migration sJAM-C = soluble JAM-C TNF = tumor necrosis factor TLR = Toll-like receptor

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

occlusion and etiology according to the Trial of Org 10172 inAcute Stroke Treatment classification IS volumes were mea-sured on diffusion sequences acquired on a 3-Tesla MRI in-strument at admission (and not measured for patients withcontraindications to brain MRI) A semiautomatic segmenta-tion procedure by Olea Sphere postprocessing software wasused for analysis

The 22 age-matched recruited healthy controls (HCs) pre-viously presented16 with blood biochemical and hematologicvalues within the normal range were recruited from the Unit ofNeurology of Memory and Language Centre HospitalierSainte Anne (Paris France) HCs were close members of thefamily of patients followed in the memory clinic As previouslydescribed16 HCs had no memory complaint or cognitive def-icit In addition we did not include HCs with a history of IS

Exclusion criteria in the HC and patient groups included acuteor chronic inflammatory conditions and treatment with cor-ticosteroid or nonsteroidal anti-inflammatory drugs both ofwhich could modulate neutrophil functions

Whole blood was sampled kept on ice and transported im-mediately to the laboratory for neutrophil analysis Impor-tantly all patients were sampled at inclusion (day 0 D0)within the first 6 hours after IS symptom onset 14 also hadsamples taken on D2 and D7

Standard protocol approvals registrationsand patient consentsThe Ethics Committee of Saint-Antoine Hospital approvedthe study and all patients or their proxies provided writteninformed consent before participating began The HCs wererecruited as part of their participation in the prospectivelongitudinal IMABio3 study (PHRC-0054-N 2010) whichwas approved by the Institutional Review Committee of Pitie-Salpetriere Hospital

Determination of surface molecule expressionon resting neutrophilsHeparinized whole-blood samples (500 μL) were kept on iceand stained with allophycocyanine (APC)-anti-humanCD62L (BD Biosciences San Jose CA) and PE-anti-humanCD11b (clone ICRF44 BD Biosciences) antibodies as pre-viously reported15 The senescent CXCR4brightCD62Ldim

neutrophil subset and the immunosuppressive CD16brightCD62Ldim neutrophil subset were investigated as previouslydescribed16 To investigate the rTEM neutrophil subsetsamples kept on ice were incubated for 45 minutes withFITC-anti-human CD81 (BD Biosciences) and PE-anti-CD54 (BD Biosciences) antibodies The red blood cells werethen lysed with BD FACS lysing solution and resuspendedwith Cell Fix (BD Biosciences)16

Measurement of neutrophil oxidative burstSuperoxide anion (O2

minus) production by neutrophils wasmeasured with a flow cytometryndashbased assay derived from the

hydroethidine (HE) oxidation technique as previously de-scribed16 After loading with HE for 15 minutes whole-bloodsamples were incubated for 45 minutes at 37degC with phos-phate buffered saline (PBS) or either of the following Toll-likereceptor (TLR) agonists Pam3CSK4 or lipopolysaccharide(LPS) or tumor necrosis factor (TNF)-α as previouslyreported22 Samples were then treated with PBS or 10minus6 MN-formylmethionyl-leucyl-phenylalanine (fMLP) for 5minutes22 Erythrocytes were lysed before analysis by flowcytometry

Measurement of neutrophil apoptosisand necrosisSpontaneous neutrophil cell death in whole blood wasquantified with annexin V and the impermeant nuclear dye7-amino-actinomycin D (7-AAD) Whole-blood samples(500 μL) were incubated with APC-anti-CD15 antibody(clone HI98 BD Biosciences) FITC-annexin V and 7-AAD(BD Biosciences) as previously described and analyzed byflow cytometry16

Flow cytometry analysisCells were analyzed with a Gallios flow cytometer and the datawith Kaluza software (Beckman Coulter) To determineneutrophil expression of surface molecules and ROS pro-duction forward and side scatter were used to identify thegranulocyte population and to gate out other cells and debrisThe purity of the gated cells was assessed using FITC- or PE-conjugated CD3 CD45 CD14 and CD15 antibodies Tenthousand events were analyzed per sample and the fluores-cence pulses were amplified by 4-decade logarithmic ampli-fiers In all cases unstained cells were run and thephotomultiplier settings were adjusted so that the unstainedcell population appeared in the lower left-hand corner of thefluorescence display In the multicolor analysis single-cellcontrols were used to optimize signal compensation All theresults were obtained with the use of a constant photo-multiplier gain value To measure cell death in whole blood byflow cytometry neutrophils were identified as CD15high cellsand 2 times 105 events were analyzed per sample

Measurement of intravascular NETosisNETs are networks of DNA histone and proteolytic enzymesproduced by activated neutrophils including myeloperox-idase (MPO) citrullinated histone 3 and elastase SerumNET levels were quantified by detecting MPO-DNA com-plexes in serum samples To prepare the DNA standardNETs from a healthy subject were produced and isolated andDNA was quantified in NET samples with PicoGreen NET-associated MPO-DNA complexes were then quantified alsoas previously described17

Measurement of soluble pro- andanti-inflammatory mediatorsWhole-blood samples were centrifuged for 15 minutes at1000gwithin 30minutes of collection Soluble chemokine (IL-8) and cytokines (IL-1β IL-6 TNF-α G-CSF IL-18 IL-22

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 3

IL-10 and transforming growth factor-β) were detected withLuminex assays (Luminex Performance Assays RampD Sys-tems)High-mobility group box-1 (HMGB1) and elastase weredetected from serum by ELISA

StatisticsStatistical analyses were performed with Rstudio 10143software All tests were 2 tailed with a significance level of α =005 When a parametric test was used normality of distri-bution was tested with the Shapiro-Wilk test Differencesbetween groups were assessed with the χ2 test or analysis ofvariance (ANOVA) followed by the Tukey post hoc test asappropriate ANOVA adjusted for age was used to compareneutrophil markers between the IS groups and controls (withage as a covariate) Bonferroni correction was used for mul-tiple comparisons Linear partial correlation analysis withadjustment for covariates identified correlations

Data availability statementData not provided will be available upon request

ResultsParticipantsrsquo characteristicsThe table summarizes the clinical characteristics of the 41patients with IS and 22 HCs Patients were included in 2steps the results from the first set of 27 patients led us toinclude a second set of 14 patients for complementary anal-yses The 2 patient cohorts did not differ significantly forbaseline characteristics including risk factors Patients with ISand HCs did not differ significantly for age or sex but riskfactors were significantly more frequent in the IS than theHCs The proportion of etiologies for cerebral infarctionobserved in patients appears similar to recent data18 Weobserved 4 brainstem strokes (98) 13 cortical strokes(317) 11 subcortical strokes (268) and 13 cortical andsubcortical strokes (317) Seven patients had hemorrhagictransformation Five patients died within 7 days of onset Asexpected19 patients with IS had higher polymorphonuclearneutrophil (PMN) counts than HCs and higher PMNlymphocyte ratios

Highly activated circulating neutrophils inpatients with acute strokeWe analyzed neutrophil activation in the 27 patients with ISincluded in the first set of patients and in the 22 HCs Neu-trophil activation is associated with the modulation of surfacemolecules ie decreased L-selectin (CD62L) and increasedβ2-integrins (ie CD11bCD18) through either stimulus-induced shedding or translocation from intracellular granulesAs figure 1 A and B show resting neutrophils from patientson D0 expressed less CD62L and more CD11b than HCsBecause neutrophil degranulation can also result in rapid re-lease of neutrophil serine proteases from azurophilic granulesinto the extracellular medium and circulation we measuredthe level of circulating neutrophil elastase Consistently with

previous data20 serum neutrophil elastase levels were signif-icantly higher in patients with IS than HCs (figure 1C)

Measuring ROS production by neutrophils we observed itwas higher in unstimulated neutrophils from whole blood(PBS-incubated sample) from patients than fromHCs (figure1D) We then assessed whether the higher constitutive ROSproduction by patient neutrophils was associated with higherproduction capacity in response to various activating stimuliWe have previously reported that neutrophils in whole bloodproduce minimal ROS in response to a single stimulus 16Wetherefore studied neutrophil oxidative bursts in response tothe bacterial peptide fMLP after priming with TNF-α orvarious TLR agonists ROS production by nonprimed neu-trophils (sample preincubated with PBS and stimulated withfMLP) was significantly higher in patients than in HCs (figure1E) and under the different priming conditions the signifi-cantly higher ROS production in patients with IS (figure 1E)demonstrates their neutrophil hyperreactivity

Among the patients included in our study 14 were alsosampled on D2 and D7 Except for CD62L expression whichreturned to normal levels on those days no markers of neu-trophil activation were modulated from D0 during the courseof acute IS (figure 1 FndashJ) No significant differences amongany of the neutrophil activation markers were observed be-tween patients with or without subsequent stroke-associatedinfections or patients with or without IS risk factors (suchas hypertension diabetes and hyperlipidemia) (data notshown)

These results reflect basal hyperactivation of patientsrsquo circu-lating neutrophils during acute IS associated with their hy-perreactivity to various stimuli by ROS production

Deregulation of neutrophil death in patientswith acute strokeNeutrophils constitutively undergo apoptosis and this process iscritical for the resolution of inflammation Neutrophil death bynecrosis inversely induces the release of cytotoxic mediators thatcan damage adjacent healthy tissue Measuring the percentage ofneutrophils undergoing spontaneous apoptosis and necrosisshowed that the percentage of apoptotic neutrophils at D0 wassignificantly lower in patients with IS and tended to return tonormal values during the course of IS (figure 2 A and B) Thepercentage of necrotic neutrophils however was rising atD0 andthe increase became significant fromD2 (figure 2 C andD) Thisderegulation of the balance between apoptosis and necrosiscombined with basal neutrophil hyperactivation may play a rolein establishing inflammatory processes in these patients

Neutrophils can undergo NETosis an alternative form ofprogrammed cell death associated with the release of decon-densed chromatin lined with granular components that cre-ates fibrous structures serving as NETs These NETs canentangle pathogens but also contribute to both venous andarterial thrombosis21 Patients with IS had lower levels of

4 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

circulating NETosis-derived products in their blood samplesat inclusion than HCs (figure 2E) This difference persisted atD2 and D7 (figure 2F)

Patients with or without either infection or IS risk factors didnot differ significantly for neutrophil apoptosis necrosis orintravascular NETosis (data not shown)

Expansion of the overactive senescentneutrophil subset in patientswith acute strokeThe phenotypes and functions of neutrophils change over timefrom their release into the blood (as fresh neutrophils) until theirdisappearance from the circulation (as aged neutrophils) Aged orsenescent CXCR4high neutrophils express lower levels of CD62Land higher levels ofβ2-integrins and produceROS at a higher rate

Table Characteristics of patients with ischemic stroke and HCs participating in the study

HC (n = 22)Patient set 1(n = 27)

Patient set 2(n = 14)

pValue set 1vs HC

pValue set 2vs HC

p Value set 1vs set 2

Sex (male) (n) [] 6 [353] 14 [518] 8 [5714] 036 029 099

Age yr (mean) [minndashmax] 681 [22ndash89] 726 [18ndash96] 711 [40ndash92] 020 054 059

History of (n) []

Diabetes 1 [454] 6 [222] 1 [589] 011 099 069

High blood pressure 2 [118] 21 [778] 10 [714] 0001 0001 071

Dyslipidemia 4 [235] 14 [519] 2 [143] 002 099 069

Myocardial infarction 0 9 [333] 4 [286] 0002 002 099

Atrial fibrillation 0 8 [296] 4 [286] 0006 002 099

Stroke 0 6 [222] 1 [714] 0026 039 039

NIHSS D0 (mean) [minndashmax] mdash 915 [0ndash21] 864 [1ndash23] mdash mdash 064

EtiologiesTOAST (n) []

Thromboembolism mdash 6 [222] 2 [143] mdash mdash 069

Cardioembolism mdash 11 [407] 4 [286] mdash mdash 051

Lacunar stroke mdash 1 [37] 2 [143] mdash mdash 054

Other specific cause mdash 3 [74] 3 [214] mdash mdash 064

Undetermined cause mdash 6 [222] 3 [214] mdash mdash 099

Occlusion at inclusion (n) [] mdash 16 [593] 6 [428] mdash mdash 051

Volume of cerebral infarct (mL) (mean)[minndashmax]

mdash 2392[004ndash2408]

1617 [0ndash638] mdash mdash 099

Treatment at acute stage (n) [] mdash

IV Thrombolysis mdash 17 [629] 5 [357] mdash mdash 0115

Endovascular treatment mdash 9 [333] 4 [286] mdash mdash 099

Time from symptom onset to blood sample(min) (mean) [minndashmax]

mdash 129 [45ndash240] 186 [95ndash340] mdash mdash 009

Previous treatment by statins 2 [117] 9 [333] 3 [214] 0158 0636 0494

Previous treatment by antithromboticagents

0 8 [296] 4 [286] 0006 0017 099

Neutrophil count (GL) (mean) [minndashmax] 315[232ndash480]

61[237ndash1440]

785[233ndash153]

0007 0002 022

Lymphocyte count (GL) (mean) [minndashmax] 168[106ndash215]

148[053ndash315]

152[057ndash326]

043 055 091

Ratio PMNlymphocyte (mean) [minndashmax] 209[137ndash436]

563[104ndash2334]

770[104ndash2684]

0023 0024 040

Abbreviations HC = healthy control NIHSS = NIH Stroke Scale TOAST = Trial of Org 10172 in Acute Stroke TreatmentSex risk factors and type of treatment were compared with the χ2 test The Mann-Whitney test was used to compare quantitative variablesWe considered all thrombolyses and all mechanical thrombectomies

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 5

than the total circulating pool8 Conversely in contexts of acuteinflammation9 and healthy aging22 an immunosuppressive subsetof CD16brightCD62Ldim neutrophils show decreased adhesionproperties and less ROS production Changes in the phenotypicand functional features of patientsrsquo neutrophils might thus berelated to an imbalance between these 2 neutrophil subsets Thesenescent CXCR4brightCD62Ldim neutrophil subset expandedin patients with IS at D0 (figure 3 A and C) whereas the per-centage of neutrophils in the CD16brightCD62Ldim

immunosuppressive subset did not differ between patientswith IS and HCs (figure 3 B and D) Accordingly patientswith IS had a higher ratio of senescent to immunosuppressivesubsets than HCs (figure 3 E) Similar results were observedin patients with IS at D2 and D7 (figure 3 FndashH) MoreoverPMN subsets did not differ significantly between patients withor without infection or IS risk factors (data not shown) Theseresults reflect the expansion of the senescent subset in patientswith IS may lead to greater PMN oxidative burst

Figure 1 Hyperactivation of circulating neutrophils from patients with ischemic stroke

(AndashE) Adhesion molecules expres-sion polymorphonuclear neutrophil(PMN) elastase and ROS productionwere studied in patients with ISat inclusion Expression of CD62L(A) and CD11b (B) at the surfaceof resting PMNs was studied onwhole-blood samples kept on ice andincubeted with anti-CD62L and anti-CD11b monoclonal antibodiesResults are expressed as mean fluo-rescence intensity (MFI) (C) Circulat-ing levels of PMN elastase werequantified by ELISA results areexpressed as ngmL (D and E) ROSproduction by unstimulated PMNswas studied after treatment of whole-blood samples for 50 minutes withPBS results are expressed as MFI (D)ROS production by stimulated PMNswas measured after pretreatment ofwhole-blood samples for 45 minuteswith PBS Pam3CSK4 (TLR12 agonist1 μgmL) or LPS (TLR4 agonist 10 ngmL) or TNF-α (TNF 5 ngmL) and in-cubation for 5 minutes with fMLP(10minus6 M) results are expressed asMFI(E) (FndashJ) Follow-up of surface expres-sion of CD62L (F) surface expressionof CD11b (G) circulating levels ofPMN elastase (H) and ROS pro-duction by unstimulated (I) and stim-ulated PMNs (J) during the first weekafter IS symptoms All samples camefrom age-matched HCs (n = 22) andpatients with IS at day 0 (n = 27) day 2(n = 14) and day 7 (n = 14) Values aremean plusmn SEM Significantly differentfrom controls p lt 005 p lt 001 plt 0001 adjusted for age D0 = day 0D2 = day 2 D7 = day 7 fMLP = N-for-mylmethionyl-leucyl-phenylalanineHC = healthy control IS = ischemicstroke LPS = lipopolysaccharide ROS =reactive oxygen species SEM = stan-dard error of mean TLR = Toll-like re-ceptor TNF = tumor necrosis factor

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

Increased levels of soluble JAM-C wereassociated with an increase in the rTEMneutrophil subset in patients withacute strokeJAM-C is an adhesion molecule expressed by endothelialcells that plays a role in tight junction formation leukocyteadhesion and transendothelial migration Elastase-inducedproteolytic cleavage of endothelial JAM-C leading to sol-uble JAM-C (sJAM-C) was recently reported to be in-strumental in promoting neutrophil rTEM in vivo23

Measuring the circulating levels of JAM-C in our first co-hort of 27 patients with IS showed a higher level at D0 thanin HCs (figure 4A) values returned to normal at D2 (figure4B) As expected23 increased sJAM-C levels were

associated with an increased circulating level of neutrophilelastase (figure 4C)

We thus performed a complementary study in a second co-hort of 14 patients with IS to investigate the percentage ofcirculating rTEM neutrophils and its association with thesJAM-C level The clinical characteristics of this second co-hort did not differ from that of the main cohort (table) nordid their sJAM-C level on D0 (421 plusmn 20 pgmL and 463 plusmn 49pgmL for the patient set 1 and set 2 respectively p = 077)which was similarly significantly higher than in HCs As pre-viously reported23 a small population of neutrophils with anrTEM phenotype (CD54high CXCR1low) was consistentlyfound in HCs equivalent to 1 of all circulating neutrophilsIn patients with IS we could also identify a distinct populationof cells with an rTEM phenotype its percentage was signifi-cantly higher in patients than in HCs (figure 4 D and E)and the increase of this percentage was associated with in-creased levels of sJAM-C (figure 4F) Taken together theseresults demonstrate the expansion of the rTEM subset in ISmigration

Neutrophil abnormalities are associated withthe severity of acute ISWe then investigated whether the neutrophil abnormalitiesobserved in patients with IS might be associated with strokeseverity Patients were dichotomized according to the NIHSSscore at inclusion into minor-to-moderate (NHISS scorele12) and severe stroke (NHISS score gt12) categories Basalneutrophil activation at baseline was greater in severe than inminor-to-moderate strokes as shown by patientsrsquo lowerCD62L expression (figure 5A) higher circulating neutrophilelastase levels (figure 5C) and higher neutrophil productionof ROS (figure 5D) although CD11b expression did notdiffer significantly between the groups (figure 5B) Thesephenotypic and functional properties of neutrophils from thesevere stroke group were associated with a higher percentageof neutrophils in the senescent hyperactive subset (figure 5E)but not in the immunosuppressive subset (figure 5F) Finallythe sJAM-C level was higher in the severe stroke group (figure5G) In contrast the percentage of apoptotic (figure 5H) andnecrotic neutrophils (data not shown) and the intravascularNETosis level (data not shown) did not differ between thepatient groups Higher NIHSS scores at D0 were associatedwith decreased expression of CD62L (figure 5I) increasedROS production by both unstimulated (figure 5J) and LPS-primed neutrophils (figure 5K) and an increase in the per-centage of the senescent hyperactive subset (figure 5L) thelevel of sJAM-C (figure 5M) and the percentage of rTEMneutrophils (figure 5N) Increased NIHSS score at D2 was alsoassociated with increased ROS production by LPS-primedneutrophils (data not shown) The correlations were adjustedfor age We found no correlation between neutrophil parame-ters and the infarct volume at inclusion and the presence ofmacroangiopathy the occurrence of hemorrhagic trans-formation the use of antithrombotic treatment or the use ofthrombolysis or endovascular treatment (data not shown)

Figure 2 Deregulation of neutrophil death in patients withischemic stroke

(AndashD) Polymorphonuclear neutrophil (PMN) death was measured immedi-ately after sampling by staining with annexin V and 7-AAD Results areexpressed as the percentages of apoptotic (A and B) and necrotic (C and D)PMNs (A and C) Values of PMN death in patients with IS at inclusion (B andD) Follow-up of PMN death during the first week after IS symptoms (E and F)Circulating NETs were quantified by MPO-DNA complex ELISA results areexpressed as ngμL DNA Values of circulating NETs in patients with IS atinclusion (E) Follow-up of intravascular NETosis during the first week after ISsymptoms (F) All samples came from age-matched healthy controls (HCs n= 22) and patients with IS at day 0 (n = 27) day 2 (n = 14) and day 7 (n = 14)Values aremean plusmn SEM Significantly different from controls p lt 005 p lt001 p lt 0001 adjusted for age AAD = amino-actinomycin D D0 = day 0D2 = day 2 D7 = day 7 HC = healthy control IS = ischemic stroke MPO =myeloperoxidase NET = neutrophil extracellular trap SEM = standard errorof mean

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 7

Considered together these results strongly suggest that theimpaired homeostasis of circulating neutrophils associatedwith neutrophil hyperactivation is correlated with the clinicalseverity of acute stroke disease progression

Proinflammatory environment in patientswith acute strokeIschemic cerebral tissue rapidly induced the release ofcytokines chemokines and danger-associated molecularpatterns (DAMPs) including HMGB124 Because cyto-kines25 and HMGB126 strongly regulate neutrophil activitywe investigated patientsrsquo peripheral proinflammatory envi-ronment Levels of classic proinflammatory (IL-1β IL-6IL-8 and TNFα) and anti-inflammatory (IL-10 and trans-forming growth factor-β) cytokines were similar in the IS

patient and HC groups (figure 6A) Serum levels of G-CSFand IL-22 a bifunctional cytokine with both proin-flammatory and protective functions were significantlylower in the IS than the HC group Inversely serum levels ofthe proinflammatory cytokine IL-18 were higher in thepatients with IS (figure 6A) as were circulating HMGB1levels (figure 6 B and C) which were associated with theclinical NIHSS score on D0 (figure 6D) Taken togetherthese results demonstrate higher circulating levels of theproinflammatory cytokine IL-18 and of the DAMPHMGB-1in patients with IS

We then investigated correlations between neutrophilparameters and proinflammatory mediator levels Neutro-phil markers were not correlated with cytokine levels In

Figure 3 Impaired homeostasis of the circulating neutrophils in patients with ischemic stroke

(AndashE) Analysis of senescent and immunosuppressive polymorphonuclear neutrophil (PMN) subsets in patientswith IS at inclusionWhole-blood sampleswereincubated for 45minutes at 4degC with Pe-Cy7-anti-human CXCR4 PE-anti-human CD11b and APC-antihuman CD62L (A and B) or with FITC-anti-human CD16PE-anti-human CD11c Pe-Cy7-antihuman CD11b and APC-anti-human CD62L (C and D) antibodies (A) Representative dot plots of the PMN phenotypeaccording to CXCR4 and CD62L expression in aHC (left) and a patient with IS (right) (B) Representative dot plots of the PMNphenotype according to CD16 andCD62L expression in an HC (left) and a patient with IS (right) (C) Percentages of the CXCR4brightCD62Ldim senescent PMN subset (D) Percentages of theCD16brightCD62Ldim immunosuppressive PMN subset (E) Ratio between the senescent and the immunosuppressive PMN subsets (FndashH) Follow-up ofsenescent PMN subset (F) immunosuppressive PMN subset (G) and ratio between the senescent and the immunosuppressive PMN subsets (H) during thefirst week after IS symptoms All samples came fromage-matchedHCs (n = 22) andpatientswith IS at day 0 (n = 27) day 2 (n = 14) andday 7 (n = 14) Values aremeanplusmn SEM Significantly different from controls p lt 005 p lt 001 p lt 0001 adjusted for age D0 =day 0 D2 = day 2 D7 = day 7 HC = healthy control IS= ischemic stroke SEM = standard error of mean

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

contrast the increased serum HMGB1 level was associatedwith decreased CD62L expression on resting neutrophils(figure 6E) increased CD11b expression (figure 6F) andincreased ROS production by primed neutrophils (figure6G) These findings suggest that HMGB1 might preactivateneutrophils in vivo

DiscussionIn the present study phenotypes and functions of peripheralneutrophils from patients less than 6 hours after IS have beenextensively investigated Our results demonstrated continu-ous basal hyperactivation of circulating neutrophils duringacute IS characterized by lower L-selectin expression andhigher CD11b expression at the cell surface increased ROSproduction by neutrophils and greater circulating levels ofneutrophil elastase The release of this soluble cytotoxicmolecule might induce vascular damage that aggravates is-chemia or diffuses into the brain parenchyma and causes

tissue injury Neutrophil hyperactivation was associated withderegulation of the equilibrium between apoptotic and ne-crotic PMNs which may be a supplemental factor promotingacute ischemic inflammation Furthermore the lack of dif-ference in neutrophil changes according to the presence orabsence of risk factors for stroke such as type 2 diabetes andhypertension suggests that these abnormalities might be di-rectly linked to IS

Activated neutrophils may contribute to vascular inflammationduring IS through neutrophil-induced BBB impairment TheBBB forms a protective barrier around the brain with the es-sential function of maintaining brain homeostasis During is-chemia and subsequent reperfusion its integrity is disruptedwhich results in the leakage of blood-borne proteins into braintissue Loss of BBB integrity correlates with poorer long-termoutcomes in patients with IS27 ROS including those producedextracellularly by neutrophils28 have been reported to induceBBB breakdown29 In particular ROS activate matrix

Figure 4 Patients with ischemic stroke have higher levels of soluble JAM-C and of PMN reverse transmigration

(A and B) sJAM-C was quantified byELISA results are expressed as pgmL(A) Values of sJAM-C in age-matchedHCs (n = 22) and patients with IS at in-clusion (n = 27) (patients included in set1) (B) Follow-up of sJAM-C levels duringthe first week after IS symptoms in 10patients (C) Correlation between thelevels of sJAM-C and of circulating PMNelastase (D and E) Quantification ofrTEM PMNs in patients with IS includedin set 2 Whole-blood samples wereincubated for 45 minutes at 4degCwith FITCanti-human CD181 and PE-anti-human CD54 antibodies (D)Representative dot plots of the PMNphenotype according to CXCR4 andCD62L expression in an HC (left) anda patient with IS (right) (E) Percentagesof the rTEM PMN subset (CD54highCXCR1low) in age-matched HCs (n = 17)and patients with IS at inclusion (n = 14)(F) Correlation between the percentageof rTEM PMN subset and the level ofsJAM-C in patients with IS included inthe set 2 Significantly different fromcontrols p lt 005 p lt 001 p lt0001 adjusted for age D0 = day 0 D2 =day 2 D7 = day 7 HC = healthy controlIS = ischemic stroke PMN = poly-morphonuclear neutrophil rTEM = re-verse transendothelialmigration sJAM-C=soluble JAM-C

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 9

metalloproteinase (MMP) activation which in turn plays a keyrole in BBB breakdown by disrupting the basement membraneof the neurovascular unit30 Among the variousMMPsMMP-9(gelatinase B) is thought to be central to BBB impairmentNeutrophils are the major source of MMP-9 after IS and

neutrophil-derivedMMP-9 has been shown to be important inpostischemic BBB disruption leukocyte infiltration and braindamage29 BBB disruption triggers inflammatory response andinfiltration of immune cells including neutrophils which are inturn associated with increased neuronal cell death through the

Figure 5 Neutrophil hyperactivation is associated with disease severity

(A andB) Expression of CD62L (A) and CD11b (B) at the surface of resting polymorphonuclear neutrophils (PMNs) Results are expressed asMFI (C) Circulatinglevels of neutrophil elastase were quantified by ELISA (D) ROS production by unstimulated PMNs results are expressed in MFI (E) Percentages of theCXCR4brightCD62Ldim senescent PMN subset (F) Percentages of the CD16brightCD62Ldim immunosuppressive PMN subset (G) sJAM-C was quantified byELISA results are expressed as pgmL (H) PMN apoptosis was measured immediately after sampling by staining with annexin V and 7-AAD Results areexpressed as percentages of apoptotic PMNs All measurements came from patients withminor-to-moderate stroke (IS NHISS le 12 n = 17) and patients withsevere stroke (IS NHISS gt 12 n = 10) at the inclusion Values aremean plusmn SEM Statistical significance as determined by the nonparametricMann-Whitney test isindicated Significantly different p lt 005 p lt 001 p lt 0001 Correlation between the NIHSS score at inclusion and CD62L expression at the PMNsurface (I) ROS production by unstimulated PMNs (J) ROS production by LPS-stimulated PMNs (K) percentage of the CXCR4brightCD62Ldim senescent PMNsubset (L) sJAM-C level (M) and percentage of rTEM PMNs (N) AAD = amino-actinomycin D D0 = day 0 IS = ischemic stroke LPS = lipopolysaccharide MFi =mean fluorescence intensity NIHSS =NIH Stroke Scale PMN = polymorphonuclear neutrophil ROS = reactive oxygen species SEM = standard error ofmeansJAM-C = soluble JAM-C

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

release of neurotoxic substances and secondary injuries Assenescent neutrophils in the circulation have been reported torapidlymigrate to the inflammatory site31 we speculate that theexpansion of the senescent neutrophil subset observed inpatients with IS might result in enhanced migration of thesehyperactive cells in the brain Both the percentage of senescentneutrophils and the parameters of neutrophil hyperactivationwere correlated with disease severity measured by the NIHSSscoreWe did not however find any correlationwith the infarctvolume at inclusion In fact a limitation of our study is related

to the fact that themeasured volumematches the ischemic coreand does not include the volume of potentially reversible ischemia(penumbra) It has been suggested that inflammatory processestake place in the penumbral area and contribute to potentialenlargement of the infarct32 Furthermore themeasured volumeson diffusion do not match with the final volume especiallyin treated patients3334 A potential association between neutro-phil abnormalities and the progression of damage in the pen-umbra and the final ischemic volume have to be explored infurther studies In addition we had a relatively limited number of

Figure 6 Proinflammatory mediators in patients with ischemic stroke

(A) Circulating levels of pro- and anti-inflammatory cytokines weremeasured with Luminex assays in samples from controls (HCs n = 22) and patients with ISat day 0 (n = 27) (B and C) Circulating levels of HMGB1 were measured in serum by ELISA (B) Comparative analysis of circulating levels of HMGB1 betweenage-matched HCs (n = 22) and patients with IS (IS) at day 0 (n = 27) (C) Follow-up of circulating levels of HMGB1 in patients with IS at day 0 2 and 7 Values aremean plusmn SEM Significantly different from controls p lt 005 p lt 001 p lt 0001 adjusted for age Correlation between circulating levels of HMGB1 at day0 and the NIHSS score at day 0 (D) (EndashG) Correlation between circulating levels of HMGB1 at day 0 and PMN activation markers at day 0 PMN surfaceexpression of CD62L (E) and of CD11b (F) and ROS production by LPS-stimulated PMNs (G) D0 = day 0 D2 = day 2 D7 = day 7 HC = healthy control HMGB =high-mobility group box IS = ischemic stroke LPS = lipopolysaccharide NIHSS = NIH Stroke Scale PMN = polymorphonuclear neutrophil ROS = reactiveoxygen species SEM = standard error of mean

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 11

patients and our observations must be corroborated in largercohorts

Despite neutrophil hyperactivation intravascular NETosisdecreased in patients with IS starting at D2 after IS Thisdiscrepancy with previous data35 might be related to the dif-ferent techniques used to study NETosis As chromatinconstitutes the backbone of NETs and MPO is present in theNETs we adopted complexes of nucleosomes and MPO asthe definition of NET remnants as suggested17 and quanti-fied serum NET levels by detecting the MPO-DNA com-plexes The mixing of the nuclear components with thecontent of azurophilic granules is a critical step in NETosisand enables the formation of complexes between nucleo-somes andMPO In contrast in a previous study performed inpatients with IS35 3 markers were evaluated plasma cell-freeDNA nucleosomes and citrullination of histone 3 all onlyindirect markers of NETs The decrease in intravascularNETosis observed in our study could be due to the decreasedcapacity of stimulated PMNs to produce NETs11 and to thepresence of NETs in thrombotic occlusions from patientswith IS36

The occurrence of neutrophil reverse transmigration afterischemiareperfusion injury has previously been reported inanimal models23 Our findings describe an increased per-centage of neutrophils with an rTEM (CD54highCXCR1dim)phenotype in human IS The recent report that rTEM neu-trophils show an activated morphology with unaltered anti-microbial activity suggests that they might be deleterious ifreinfiltrated into remote organs37 Moreover rTEM neu-trophils are relatively rigid a physical characteristic that mightdelay their passage through the tissue microvasculature andprolong contact with the sinusoids These cells could becomemechanically entrapped in the microvasculature of majororgans (most likely in the pulmonary circulation) We thuspostulate that a larger population of functionally primedrecirculating neutrophils in patients with IS with a pre-dilection for mechanical or adhesive entrapment in the vas-culature could contribute to distant organ damage andmultiorgan failure As these rTEM neutrophils have beencharacterized by their prolonged survival and enhanced oxi-dative burst38 their expansion in patients with IS might ex-plain at least in part the higher ROS production and thelower rate of neutrophil apoptosis in our cohort Furthermorebecause rTEM neutrophils are reported to inhibit T-cellproliferation9 we can speculate that they might contribute tothe T lymphopenia observed during the stroke-inducedimmunodepression phase

Consistent with data suggesting that sJAM-C may serve as anindirect marker of neutrophil rTEM2338 higher levels of thisadhesion molecule in patients with IS associated with thepercentage of rTEM neutrophils On the other hand sJAM-Calso acts as a potent proangiogenic molecule with chemotacticaction for endothelial cells and it induces their formation ofvascular tubes in vitro39 After ischemic injury angiogenesis

increases the number of new collateral conduits enhancingthe blood supply and improving ischemic brain function40

Despite this potential beneficial action of sJAM-C we clearlyobserved that the sJAM-C level was higher in the severe strokegroup

As previously reported4142 we observed higher circulatinglevels of the proinflammatory cytokine IL-18 and of theDAMP HMGB-1 in patients with IS than in age-matchedHCs IL-18 production requires activation of the inflamma-some a group of multimeric protein complexes that sensestress signals activate caspase-1 and thus cause its proteolyticcleavage Notably IS increases the expression and activationof the Nod-like receptor (NLR) pyrin domain containing 1and 3 (NLRP1 and NLRP3) inflammasome proteins andboth IL-1β and IL-18 in neurons43 IL-1844 and HMGB126

enhance neutrophil functions including priming neutrophilsfor oxidative burst and degranulation In turn activated neu-trophils produce IL-18 In addition necrotic cells includingnecrotic neutrophils passively release HMGB1 Of interestwe found that the percentage of necrotic neutrophils washigher in patients with IS than HCs These processes togethermight thus maintain a vicious cycle We also observed thatincreased plasma levels of HMGB-1 were associated withmarkers of neutrophil activation

The phenotypic and functional neutrophil changes that ac-company acute IS may also be related to the expansion of theoveractive aged neutrophil subpopulation A recent studyshowed that neutrophil aging is regulated by the microbiota8

Recent studies report gut dysbiosis in patients with IS4546

Commensal microbiota affects IS outcome in mice with is-chemic brain injury by impairing immune homeostasis in thesmall intestine thus increasing IL-17+ γδ T cells and de-creasing regulatory T cells (Tregs)47 Of note γδ T cellsa major lymphocyte population with innate immune featurescan aggravate ischemic brain injury by secreting IL-17 andgenerating chemotactic signals for peripheral myeloid cellssuch as neutrophils48 In contrast Tregs are known to inhibitneutrophil functions49 in rodent IS models adoptivelytransferred Tregs exert a protective effect by modulating pe-ripheral neutrophils and thus prevent proteolytic damage tothe BBB50 Alterations in the microbiota could contributeboth to expansion of senescent neutrophils and changes inneutrophil activation in patients with IS We can speculatethat modulation of the microbiome might affect diseaseprogression by limiting neutrophil aging and reduction pro-duction of neutrophil-derived cytotoxic molecules

We demonstrated that homeostasis of circulating neutrophilsis impaired during acute IS These findings need to be repli-cated in an independent cohort of patients in which a metic-ulous definition of neutrophil subsets will be performed Theexpansion of aged neutrophils might play a role in poststrokesystemic inflammation and BBB disruption and rTEM neu-trophils might further contribute to the propagation of sys-temic inflammation Our findings highlight new potential

12 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

therapeutic approaches of stroke based on neutrophil mod-ulation in particular by rebalancing the ratio of senescent toimmunosuppressive neutrophils or decreasing reverse neu-trophil transmigration or both Further studies are neededhowever to determine whether these changes might serve asa predictive indicator of IS

Author contributionsC Elbim conceived the study designed the experiments andsupervised the project D Weisenburger-Lile and Y Dongconducted the experiments and analyzed the data DWeisenburger-Lile M Yger GF Polara and B Laperguewere involved in patient recruitment and characterization andin the data analysis G Weisenburger-Lile T Chaigneau andR Zapata Ochoa participated in the data analysis DWeisenburger-Lile S Alamowitch and C Elbim wrote themanuscript All authors read and approved the content of thefinal manuscript

AcknowledgmentThe authors thank the nursing staff of the Stroke Unit ofSaint-Antoine Hospital Pitie Salpetriere Hospital and FochHospital for the patient management The authors gratefullyacknowledge Annie Munier from the LUMIC flow cytometryfacility DWL received a grant from the ldquoSociete Francaise deNeurologierdquo for a part of this work

Study fundingNo targeted funding reported

DisclosureThe authors report no disclosures relevant to the manuscriptDisclosures available NeurologyorgNN

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationDecember 19 2018 Accepted in final form March 12 2019

References1 Ross AM Hurn P Perrin N Wood L Carlini W Potempa K Evidence of the

peripheral inflammatory response in patients with transient ischemic attack J StrokeCerebrovasc Dis 200716203ndash207

2 Neumann J Riek-Burchardt M Herz J et al Very-late-antigen-4 (VLA-4)-mediatedbrain invasion by neutrophils leads to interactions with microglia increased ischemicinjury and impaired behavior in experimental stroke Acta Neuropathol 2015129259ndash277

3 Shi Y Zhang L Pu H et al Rapid endothelial cytoskeletal reorganization enables earlyblood-brain barrier disruption and long-term ischaemic reperfusion brain injury NatCommun 2016710523

4 Perez-de-Puig I Miro-Mur F Ferrer-Ferrer M et al Neutrophil recruitment to thebrain in mouse and human ischemic stroke Acta Neuropathol 2015129239ndash257

5 Enlimomab Acute Stroke Trial I Use of anti-ICAM-1 therapy in ischemic strokeresults of the Enlimomab Acute Stroke Trial Neurology 2001571428ndash1434

6 Krams M Lees KR Hacke W et al Acute stroke therapy by inhibition of neutrophils(ASTIN) an adaptive dose-response study of UK-279276 in acute ischemic strokeStroke 2003342543ndash2548

7 Gunzer M Traps and hyper inflammationmdashnew ways that neutrophils promote orhinder survival Br J Haematol 2014164189ndash199

8 Zhang D Chen G Manwani D et al Neutrophil ageing is regulated by the micro-biome Nature 2015525528ndash532

9 Pillay J Kamp VM van Hoffen E et al A subset of neutrophils in human systemicinflammation inhibits T cell responses through Mac-1 J Clin Invest 2012122327ndash336

10 de Oliveira S Rosowski EE Huttenlocher A Neutrophil migration in infection andwound repair going forward in reverse Nat Rev Immunol 201616378ndash391

11 Ruhnau J Schulze K Gaida B et al Stroke alters respiratory burst in neutrophils andmonocytes Stroke 201445794ndash800

12 Kelly PJ Morrow JD NingM et al Oxidative stress andmatrix metalloproteinase-9 inacute ischemic stroke the biomarker evaluation for antioxidant therapies in stroke(BEAT-Stroke) study Stroke 200839100ndash104

13 Pallister I Bhatia R Katpalli G Allison D Parker C Topley N Alteration of poly-morphonuclear neutrophil surface receptor expression and migratory activity afterisolation comparison of whole blood and isolated PMN preparations from normaland postfracture trauma patients J Trauma 200660844ndash850

14 Furie KL Jayaraman MV 2018 guidelines for the early management of patients withacute ischemic stroke Stroke 201849509ndash510

15 Campillo-Gimenez L Casulli S Dudoit Y et al Neutrophils in antiretroviral therapy-controlled HIV demonstrate hyperactivation associated with a specific IL-17IL-22environment J Allergy Clin Immunol 20141341142ndash1152e5

16 Dong Y Lagarde J Xicota L et al Neutrophil hyperactivation correlates with Alz-heimerrsquos disease progression Ann Neurol 201883387ndash405

17 Kessenbrock K Krumbholz M Schonermarck U et al Netting neutrophils in auto-immune small-vessel vasculitis Nat Med 200915623ndash625

18 Bejot Y Bailly H Durier J GiroudM Epidemiology of stroke in Europe and trends forthe 21st century Presse Med 201645e391ndashe398

19 Xue J Huang W Chen X et al Neutrophil-to-Lymphocyte ratio is a prognosticmarker in acute ischemic stroke J Stroke Cerebrovasc Dis 201726650ndash657

20 Vogelgesang A Lange C Blumke L et al Ischaemic stroke and the recanalization drugtissue plasminogen activator interfere with antibacterial phagocyte functionJ Neuroinflammation 201714140

21 Martinod K Wagner DD Thrombosis tangled up in NETs Blood 20141232768ndash2776

22 Sauce D Dong Y Campillo-Gimenez L et al Reduced oxidative burst by primedneutrophils in the elderly individuals is associated with increased levels of theCD16brightCD62Ldim immunosuppressive subset J Gerontol A Biol Sci Med Sci201772163ndash172

23 Colom B Bodkin JV Beyrau M et al Leukotriene B4-neutrophil elastase Axis drivesneutrophil reverse transendothelial cell migration in vivo Immunity 2015421075ndash1086

24 Shichita T Ito M Yoshimura A Post-ischemic inflammation regulates neural damageand protection Front Cell Neurosci 20148319

25 Elbim C Bailly S Chollet-Martin S Hakim J Gougerot-Pocidalo MA Differentialpriming effects of proinflammatory cytokines on human neutrophil oxidative burst inresponse to bacterial N-formyl peptides Infect Immun 1994622195ndash2201

26 Berthelot F Fattoum L Casulli S Gozlan J Marechal V Elbim C The effect ofHMGB1 a damage-associated molecular pattern molecule on polymorphonuclearneutrophil migration depends on its concentration J Innate Immun 2012441ndash58

27 Brouns R Wauters A De Surgeloose D Marien P De Deyn PP Biochemical markersfor blood-brain barrier dysfunction in acute ischemic stroke correlate with evolutionand outcome Eur Neurol 20116523ndash31

28 Ullen A Singewald E Konya V et al Myeloperoxidase-derived oxidants induce blood-brain barrier dysfunction in vitro and in vivo PLoS One 20138e64034

29 Gidday JM Gasche YG Copin JC et al Leukocyte-derived matrix metalloproteinase-9 mediates blood-brain barrier breakdown and is proinflammatory after transient focalcerebral ischemia Am J Physiol Heart Circ Physiol 2005289H558ndashH568

30 Turner RJ Sharp FR Implications of MMP9 for blood brain barrier disruption andhemorrhagic transformation following ischemic stroke Front Cell Neurosci 20161056

31 Uhl B Vadlau Y Zuchtriegel G et al Aged neutrophils contribute to the first line ofdefense in the acute inflammatory response Blood 20161282327ndash2337

32 Vidale S Consoli A Arnaboldi M Consoli D Postischemic inflammation in acutestroke J Clin Neurol 2017131ndash9

33 Luby M Warach SJ Nadareishvili Z Merino JG Immediate changes in stroke lesionvolumes post thrombolysis predict clinical outcome Stroke 2014453275ndash3279

34 Kate MP Riaz P Gioia L et al Dynamic evolution of diffusion-weighted imaginglesions in patients with minor ischemic stroke Stroke 2015462318ndash2321

35 Valles J Lago A Santos MT et al Neutrophil extracellular traps are increased inpatients with acute ischemic stroke prognostic significance Thromb Haemost 20171171919ndash1929

36 Ducroux C Di Meglio L Loyau S et al Thrombus neutrophil extracellular traps contentimpair tPA-induced thrombolysis in acute ischemic stroke Stroke 201849754ndash757

37 Ellett F Elks PM Robertson AL Ogryzko NV Renshaw SA Defining the phenotype ofneutrophils following reverse migration in zebrafish J Leukoc Biol 201598975ndash981

38 Buckley CD Ross EA McGettrick HM et al Identification of a phenotypically andfunctionally distinct population of long-lived neutrophils in a model of reverse en-dothelial migration J Leukoc Biol 200679303ndash311

39 Rabquer BJ Amin MA Teegala N et al Junctional adhesion molecule-C is a solublemediator of angiogenesis J Immunol 20101851777ndash1785

40 Fan Y Yang GY Therapeutic angiogenesis for brain ischemia a brief review J Neu-roimmune Pharmacol 20072284ndash289

41 Ormstad H Aass HC Lund-Sorensen N Amthor KF Sandvik L Serum levels ofcytokines and C-reactive protein in acute ischemic stroke patients and their re-lationship to stroke lateralization type and infarct volume J Neurol 2011258677ndash685

42 Tsukagawa T Katsumata R Fujita M et al Elevated serum high-mobility group box-1protein level is associated with poor functional outcome in ischemic stroke J StrokeCerebrovasc Dis 2017262404ndash2411

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 13

43 Fann DY Lim YA Cheng YL et al Evidence that NF-kappaB and MAPK signalingpromotes NLRP inflammasome activation in neurons following ischemic stroke MolNeurobiol 2018551082ndash1096

44 Elbim C Guichard C Dang PM et al Interleukin-18 primes the oxidative burst ofneutrophils in response to formyl-peptides role of cytochrome b558 translocation andN-formyl peptide receptor endocytosis Clin Diagn Lab Immunol 200512436ndash446

45 Singh V Roth S Llovera G et al Microbiota dysbiosis controls the neuro-inflammatory response after stroke J Neurosci 2016367428ndash7440

46 Yamashiro K Tanaka R Urabe T et al Gut dysbiosis is associated with metabolismand systemic inflammation in patients with ischemic stroke PLoS One 201712e0171521

47 Benakis C Brea D Caballero S et al Commensal microbiota affects ischemicstroke outcome by regulating intestinal gammadelta T cells Nat Med 201622516ndash523

48 Gelderblom M Weymar A Bernreuther C et al Neutralization of the IL-17 axisdiminishes neutrophil invasion and protects from ischemic stroke Blood 20121203793ndash3802

49 Lewkowicz P Lewkowicz N Sasiak A Tchorzewski H Lipopolysaccharide-activatedCD4+CD25+ T regulatory cells inhibit neutrophil function and promote their apo-ptosis and death J Immunol 20061777155ndash7163

50 Li P Gan Y Sun BL et al Adoptive regulatory T-cell therapy protects against cerebralischemia Ann Neurol 201374458ndash471

14 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

DOI 101212NXI000000000000057120196 Neurol Neuroimmunol Neuroinflamm

David Weisenburger-Lile Yuan Dong Marion Yger et al severity

Harmful neutrophil subsets in patients with ischemic stroke Association with disease

This information is current as of May 15 2019

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is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm