interleukin-26, preferentially produced by th17 ...il-26–treated mice compared with control mice...

ARTICLE OPEN ACCESS

Interleukin-26 preferentially produced byTH17 lymphocytes regulates CNS barrierfunctionBieke Broux PhD Stephanie Zandee PhD Elizabeth Gowing MSc Marc Charabati MSc

Marc-Andre Lecuyer PhD Olivier Tastet MSc Lamia Hachehouche MSc Lyne Bourbonniere MSc

Jean-Philippe Ouimet MSc Florent Lemaitre MSc Sandra Larouche DCS Romain Cayrol MD PhD FRCPC

Alain Bouthillier MD FRCSC Robert Moumdjian MD FRCSC Boaz Lahav BSc Josee Poirier BSc

Pierre Duquette MD FRCPC Nathalie Arbour PhD Evelyn Peelen PhDDagger and

Alexandre Prat MD PhD FRCPCDagger

Neurol Neuroimmunol Neuroinflamm 20207e870 doi101212NXI0000000000000870

Correspondence

Dr Prat

apratumontrealca

AbstractObjectiveTo investigate the involvement of interleukin (IL)-26 in neuroinflammatory processes inmultiple sclerosis (MS) in particular in blood-brain barrier (BBB) integrity

MethodsExpression of IL-26 was measured in serum CSF in vitro differentiated T helper (TH) cellsubsets and postmortem brain tissue of patients with MS and controls by ELISA quantitativePCR and immunohistochemistry Primary human and mouse BBB endothelial cells (ECs)were treated with IL-26 in vitro and assessed for BBB integrity RNA sequencing was performedon IL-26ndashtreated human BBB ECs Myelin oligodendrocyte glycoprotein35ndash55 experimentalautoimmune encephalomyelitis (EAE) mice were injected IP with IL-26 BBB leakage andimmune cell infiltration were assessed in the CNS of these mice using immunohistochemistryand flow cytometry

ResultsIL-26 expression was induced in TH lymphocytes by TH17-inducing cytokines and was upre-gulated in the blood and CSF of patients with MS CD4+IL-26+ T lymphocytes were found inperivascular infiltrates in MS brain lesions and both receptor chains for IL-26 (IL-10R2 and IL-20R1) were detected on BBB ECs in vitro and in situ In contrast to IL-17 and IL-22 IL-26promoted integrity and reduced permeability of BBB ECs in vitro and in vivo In EAE IL-26reduced disease severity and proinflammatory lymphocyte infiltration into the CNS whileincreasing infiltration of Tregs

ConclusionsOur study demonstrates that although IL-26 is preferentially expressed by TH17 lymphocytes itpromotes BBB integrity in vitro and in vivo and is protective in chronic EAE highlighting thefunctional diversity of cytokines produced by TH17 lymphocytes

These authors contributed equally to the manuscript

DaggerThese authors should be considered condashlast authors

From the Neuroimmunology Unit and Multiple Sclerosis Clinic (BB SZ EG MC M-AL OT LH LB J-PO FL SL RC BL JP PD NA EP AP) The Research Center ofthe Centre Hospitalier de lrsquoUniversite deMontreal (CRCHUM) Department of Neuroscience Faculty of Medicine Universite deMontreal Canada Hasselt University (BB) BiomedicalResearch Institute and Transnationale Universiteit Limburg School of Life Sciences Diepenbeek Belgium and Division of Neurosurgery (AB RM) Centre Hospitalier de lrsquoUniversitede Montreal (CHUM) Faculty of Medicine Universite de Montreal Canada

Go to NeurologyorgNN for full disclosures Funding information is 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 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

The abundance of immune cells and their products in CNSlesions and in the CSF of patients with multiple sclerosis (MS)together with the genetic risk conferred by immune gene vari-ants supports the concept that MS is an autoimmunedisorder1ndash5 It is strongly believed that myelin-reactive CD4+ Thelper (TH) lymphocytes are activated by an environmentaltrigger after which they cross the blood-brain barrier (BBB)invade the CNS and initiate a chronic relapsing inflammatorycascade leading to tissue damage36ndash8 These TH lymphocytesproduce cytokines such as interleukin (IL)-17 IL-22 andgranulocyte macrophage colony-stimulating factor (GM-CSF)the former 2 being involved in BBB breakdown8 and the latterstimulating CCR2+ monocytes which promote tissue damage9

IL-26 was first discovered as an inducible gene in Herpesvirussaimirindashtransformed human T lymphocytes10 but has sincebeen described as a TH17-associated cytokine that is regulatedby IL-1β IL-23 and RORγt51112 IL-26 is part of the IL-10cytokine family and binds to a heterodimeric receptor com-posed of the IL-10R2 and IL-20R1 chains which results inactivation of STAT1 and STAT31314 Although the IL26 geneis absent in rodents Ohnuma et al15 have recently demon-strated that recombinant human (rh) IL-26 can bind to andfunction through the murine receptor complex In recentyears studies regarding the role of IL-26 in various immu-nologic diseases have started to emerge15ndash20 In chronic in-flammatory diseases including Crohn disease rheumatoidarthritis chronic hepatitis C infection and chronic graft-versus-host disease IL-26 appears to play a proinflammatoryrole acting on epithelial cells monocytes natural killer cellsand fibroblasts respectively15ndash1719 It was also recently foundto be involved in antibacterial host defense1820ndash22 Howeverthe role of IL-26 inMS where TH17 lymphocytes are believedto play a major role has not yet been investigated

In the current study we hypothesized that IL-26 has aproinflammatory role and is involved in BBB disruption Wereport that the concentration of IL-26 is elevated in the serumand CSF of untreated patients with MS compared withhealthy individuals and controls respectively We furtherdemonstrate that IL-26ndashexpressing TH lymphocytes arefound within perivascular infiltrates in the brain of patientswith MS In contrast to our hypothesis we demonstrate thatthe administration of IL-26 in mice with experimental auto-immune encephalomyelitis (EAE) reduces disease severitycompared with control-treated mice This is associated with abarrier-promoting effect of IL-26 through upregulation of

tight junctionmolecules Overall our results demonstrate thatIL-26 preferentially produced by TH17 lymphocytes has abeneficial effect on barrier function during neuro-inflammation and reduces clinical and pathologic diseaseburden in sterile autoimmune CNS inflammation

MethodsEthicsBlood was obtained from patients with MS and healthy donors(HDs) after written informed consent and ethical approval(Centre Hospitalier de lrsquoUniversite de Montreal research ethiccommittee approval number BH07001) Patients were classifiedaccording to the 2010 McDonaldrsquos revised criteria for MS di-agnosis (Polman et al 2011) as previously described (Larochelleet al 2012) Other neurologic disease controls (OND) consistedof migraine glioblastoma and subjective neurologic symptomswith normal neurologic examination normal MRI and normalCSF analysis With informed consent and ethical approval(Centre Hospitalier de lrsquoUniversite de Montreal research ethiccommittee approval number BH07001) human temporal lobematerial was obtained from patients who underwent surgicaltreatment for intractable temporal lobe epilepsy and used forisolation of primary BBB endothelial cells (ECs) Human fetalbrain tissue was obtained with ethical approval (Centre Hospi-talier de lrsquoUniversite de Montreal research ethic committee ap-proval number HD07002) for isolation of astrocytes

All animal procedures were approved by the CHUM AnimalCare Committee in accordance with the guidelines of theCanadian Council on Animal Care (approval numberN15035APs)

Data availabilityData not provided in the article because of space limitationscan be shared at the request of other investigators for pur-poses of replicating procedures and results

ResultsIL-26 is preferentially expressed byTH17 lymphocytesTo address the phenotype of TH lymphocytes expressing IL-26we cultured human naive (CD45RA+) TH lymphocytes fromHDs in TH1 TH2 or TH17 differentiating conditionsWe foundthat TH17 lymphocytes expressed the highest level of IL26

GlossaryBBB = blood-brain barrier CLDN = claudins DEG = differentially expressed gene EAE = experimental autoimmuneencephalomyelitis EC = endothelial cell GM-CSF = granulocyte macrophage colony-stimulating factor GSEA = gene setenrichment analysis HDs = healthy donors IL = interleukin JAM = junctional adhesion molecule MOG = myelinoligodendrocyte glycoprotein mRNA = messenger RNA OCLN = occludin OND = other neurologic disease controls rh =recombinant humanTEER = transendothelial electrical resistance TH = T helper TJ = tight junction ZO = zonula occludens

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 6 | November 2020 NeurologyorgNN

messenger RNA (mRNA) after 3 days in culture which cor-related with expression of IL17 (figure e-1 A and B linkslwwcomNXIA295) TH1 lymphocytes also expressed IL26mRNA but only after 6 days in culture To identify the cytokine(or combination of cytokines) that induces expression ofIL26 mRNA in naive TH lymphocytes we treated naiveTH lymphocytes from HDs with TH17-inducing cytokinesdaily for 6 daysWe found that the combination of IL-1β IL-23and transforming growth factor (TGF)-β1 induced the highestlevel of IL26 mRNA expression in these lymphocytes (figuree-1C) and that IL-6 alone or in combination with other cy-tokines did not significantly affect IL26 gene expression Toaddress whether IL26 is also expressed in memoryTH lymphocytes we cultured CD45RO+TH lymphocytes fromHDs in TH1- or TH17-polarizing conditions IL-26 mRNA(figure e-1D) and protein (figure e-1E) expression was highestin TH17 lymphocytes generated from memory CD4+-

lymphocytes after 6 days in culture when compared withTH1 lymphocytes or ex vivomemory TH lymphocytes Becauseintracellular flow cytometry with commercially available anti-bodies does not work in our hands we used immunocyto-fluorescent microscopy to analyze coexpression of IL-26 withIL-17 and RORγt in TH17 differentiated lymphocytes of HDs(figure e-1F) Quantification revealed a greater enrichment ofIL-26+ cells in IL-17ndashexpressing TH lymphocytes comparedwith interferon (IFN)-γndashexpressing TH lymphocytes (figuree-1G) In addition levels of IL26 mRNA significantly corre-lated with mRNA levels of TH17 markers IL17 IL22 RORCand MCAM but not with Csf2 (figure e-1H) To investigatewhether IL-26 is primarily associated with TH17 lymphocytesand not with other immune cells we measured its levels viaqPCR in different ex vivo or activateddifferentiated immunecell subsets collected from HDs The highest IL26 mRNAlevels were detected in TH17 lymphocytes polarized frommemory TH lymphocytes (figure e-2A) whereas lower IL26mRNA levels were detected in CD8+ T cytotoxic 17(TC17) lymphocytes and TCR-stimulated memoryTH lymphocytes without addition of polarizing cytokinesB lymphocytes monocytes M1 and M2 macrophages andimmature and mature dendritic cells (DCs) did not have de-tectable IL26 mRNA levels

IL-26 is elevated in the serum and CSF ofuntreated patients with MSTo determine whether IL-26 is augmented in relevant bi-ological compartments during MS disease we measured theconcentration of IL-26 in the serum and CSF of controls anduntreated patients withMS IL-26 was significantly elevated inthe serum of untreated patients with relapsing-remitting MScompared with HDs (figure 1A) In addition IL-26 was ap-proximately twofold higher in the CSF of untreated patientswith MS compared with OND (figure 1B) Using MSpatientndashderived TH17 differentiated lymphocytes we alsofound that IL26 mRNA significantly correlated with expres-sion of IL17 IL22 RORC and MCAM (figure 1C) as pre-viously demonstrated using the blood of HDs In additionexpression of IL26 mRNA also correlated with expression of

Csf2mRNA in TH17 lymphocytes of patients with MS (figure1C) Finally immunohistofluorescence imaging of active MSbrain lesions (18 lesions 6 distinct patients) revealed in-numerable CD4+ T lymphocytes expressing IL-26 in peri-vascular infiltrates (figure 1D)

IL-26 increases BBB tightness throughupregulation of tight junction moleculesOur group previously demonstrated that the TH17-associatedcytokines IL-17A and IL-22 increase BBB permeability andfacilitate TH lymphocyte migration across the BBB8 BecauseIL-26 is preferentially expressed by TH17 lymphocytes wehypothesized that it has a similar effect on the BBB To in-vestigate whether BBB ECs can respond to IL-26 stimulationwe first assessed expression of the IL-10R2 and IL-20R1 chainsIndeed we detected both IL-26 receptor chains on primarycultures of human BBB ECs in vitro (figure 2A) as well as onCNS-ECs in situ (figure 2 B and C) Inflammatory conditionsdid not significantly affect the expression of either chain (figure2A) Further investigation of the 2 receptor chains demon-strated their spatial colocalization on ECs in situ both innormal-appearing white matter and in MS lesions (figure 2 Band C) suggesting that these cells can respond to IL-26

To assess the effect of IL-26 on human BBB ECs we in-vestigated the transcriptome of resting (control [CTL]) vsrhIL-26 treated BBB ECs In total 459 differentially expressedgenes (DEGs false discovery rate lt 01 amp |logFC| gt 025)were identified all in the 100 ngmL condition (figure e-3 Aand B linkslwwcomNXIA295) To understand whatfunctional pathways might be associated with these tran-scriptional changes a ranked gene-set enrichment analysis(GSEA) approach was used to alleviate the limitations ofsetting predefined numerical thresholds of significance toform discrete gene sets (See Methods section) Our datademonstrate that rhIL-26 treatment resulted mostly in ageneral downregulation of pathways notably those involvedin the signaling of proinflammatory cytokines tumor necrosisfactor-α IFN-γ IFN-α and IL-6 as well as in apoptosis andadipogenesis (figure 3A figure e-3C) The most significantenrichment was associated with a downregulation of oxidativephosphorylation and was consistent in both conditions (fig-ure e-3C) This suggests that IL-26 could mediate cellularmetabolism through decrease in energy production Of in-terest using the ranked approach for GSEA shows that al-though there are no DEGs in the 10 ngmL conditionaccording to predefined criteria the pathways associated withthe transcriptomic variation is consistent with the 100 ngmLcondition indicating that the response in both conditions iscorrelated We indeed observe correlation between the foldchanges calculated in each condition (R = 062 p lt 22 times10minus16) Because BBB integrity is highly dependent on theexpression of tight junction (TJ) molecules (eg junctionaladhesion molecules [JAM] claudins [CLDN] occludin[OCLN] and zonula occludens [ZO])23 we hypothesizedthat some of these molecules would also be downregulated onIL-26 stimulation Therefore we decided to focus on the

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 6 | November 2020 3

Figure 1 IL-26 is increased in the serum and CSF of untreated patients with MS and is expressed by CNS-infiltratinglymphocytes

(A) IL-26 protein levels in the serum of HDs (n = 6) and untreated patients with RRMS (MS n = 9) (B) IL-26 protein levels in the CSF of persons with OND (n = 8)and untreated patients withMS (n = 10) (C) Relative expression of IL26mRNAplotted against the relative expression of other T helper (TH)17-associated genes(IL17 IL22 Csf2 RORC andMCAM) in ex vivo CD4+CD45RO+ T lymphocytes TH1- and TH17-polarized lymphocytes (after 6 days in culture) from 5 to 18 patientswith MS mRNA expression is relative to 18S mRNA and was assessed by qPCR (D) Autopsy-derived MS CNS material was stained with Luxol Fast BlueHampE(LHE left panel) to identify lesions (dashed line) Colocalization of IL-26 (red) with CD4+ cells (green) and TOPRO-3 (blue nuclei) in MS lesions is shown (leftpanel) As a control CNSmaterial was stained with an isotype control and secondary antibody (red) or secondary antibody alone (green right panel) Imagesshown are representative of immunostainings on CNS samples from 6 patients withMS (3 tissue blocks per patient) Scale bars 25 μmData are presented asmean plusmn SEM (A and B) p lt 005 p lt 001 Statistical tests Student 2-tailed t test (A and B) and Pearson correlation (C) HDs = healthy donors IL = interleukinmRNA = messenger RNA OND = other neurologic disease controls RRMS = relapsing-remitting MS


expression profiles of specific BBB-associated TJ moleculeswithin the same transcriptome data This allowed us to observethat stimulationwith IL-26 upregulates themRNA levels ofTJP1(ZO-1) OCLN and CLDN18 while also downregulating F11R(JAM-1) levels (figure 3B) CLDN5 mRNA was below the de-tection limit Collectively these data indicate that unlike IL-17IL-26 has a unique effect on BBB ECs where it downregulatesproinflammatory pathways and upregulates TJ molecules

To confirm our observations at the protein level we performedWestern blot analysis on IL-26ndashtreated BBB ECs and observedan increase in JAM-1 protein levels compared with CTL BBBECs (figure 3C and figure e-4A linkslwwcomNXIA295 p =0053) The ZO-1 protein level did not change (figure e-4B p =0431) To confirm that JAM-1 upregulation is due to IL-26signaling via its heterodimeric receptor we pretreated BBB ECswith blocking antibodies against IL-10R2 or IL-20R1 beforerhIL-26 was added The IL-26ndashinduced upregulation in theJAM-1 level in BBB ECs was prevented by pretreatments withantindashIL-10R2 or antindashIL-20R1 (figure 3C and figure e-4A) Toinvestigate whether IL-26 has a functional effect on BBB in-tegrity we performed an in vitro permeability assay usingmodified Boyden chambers Briefly we treated primary humanBBB ECs grown to confluency on inserts with rhIL-26 andmeasured their permeability to 3- 10- and 70-kDa dextrans viaspectroscopy Of interest rhIL-26ndashtreated human BBB ECsacquired a significantly lower permeability coefficient to each ofthe dextrans compared with untreated BBB ECs (figure 3D)suggesting that IL-26 indeed has a positive effect on BBB

integrity compared with IL-17 and IL-22 Consistent with theseobservations the transendothelial electrical resistance (TEER)of human BBB ECs monolayers was also significantly and sus-tainably increased by rhIL-26 treatment to the same extent asthe positive control (astrocyte-conditionedmedium figure 3E)

IL-26 therapy reduces BBB permeabilityduring neuroinflammationAlthough mice do not express IL-26 they do express a func-tional receptor which responds to rhIL-2615 Previous studieson IL-26 have shown that results obtained frommouse modelsusing rhIL-26 provide information which is supportive of hu-man in vitro or in situ studies152425 Before investigating thecontribution of IL-26 on BBB permeability in vivo we firstsought to confirm whether rhIL-26 affects mouse BBB ECs invitro Mouse brain ECs were isolated and treated with 100 ngmL rhIL-26 and subsequent expression of TJ molecules JAM-1 ZO-1 and CLDN5 was assessed In line with our afore-mentioned observations we found that rhIL-26ndashtreated mouseBBB ECs significantly upregulated the expression of both JAM-1 and CLDN5 (figure 4A) whereas it had no effect on ZO-1(not shown) In addition rhIL-26 enhanced the organizationalstructure of the intercellular TJ strands between mouse BBBECs (figure 4A) To study the impact of IL-26 on the BBB invivo we analyzed its effect on BBB permeability in EAE miceimmunized with myelin oligodendrocyte glycoprotein(MOG)35ndash55 and treated with 200 ng rhIL-26 daily IP from day5ndash24 postimmunization In situ analysis of endogenous serumproteins extravasation into the CNS revealed that rhIL-26

Figure 2 Presence of IL-10R2 and IL-20R1 on BBB ECs

(A) IL-26 receptor expression onresting (CTL) and inflamed (IFNγ andTNFα IT) human BBB ECs by West-ern blot Representative image of n =4 experiments is shown (B) Autopsy-derived MS CNS material stained forIL-10R1 (red) and IL-20R2 (green) andwith TOPRO-3 (blue nuclei) Repre-sentative plots of IL-20R1 and IL-10R2signal intensity and colocalization ofboth receptor chain (upper graphs) inareas 1 and 2 are shown for NAWM(left) or MS lesions (right) (C) Semi-quantitative analysis of the signal in-tensity from 10 μm z-stackreconstructions along the line mark-ers in the images shown in (B) for IL-20R1 and IL-10R2 in NAWM and MSlesion Mean fluorescence intensitywas averaged from 3 distinct mea-surements on each vessel (n = 33blood vessels) Images shown arerepresentative of immunostainingson brain lesions of 6 patients with MS(1ndash2 tissue blocks per patient) Scalebars 50 μm for lesion and 30 μm forNAWM Data are represented asmean plusmn SEM (C) Statistical tests Stu-dent 2-tailed t test (C) BBB ECs =blood-brain barrier endothelial cellsCTL = control IL = interleukin NAWM= normal-appearing white matter


Figure 3 IL-26 enhances BBB function in vitro

(A and B) HumanBBB ECs derived from1donor were treatedwith 0 10 or 100 ngmL recombinant human (rh)IL-26 in triplicate for 24 hours RNA sequencingwas performed onmessenger RNA (mRNA) isolated from these samples (A) A heatmap of the enrichment scores for pathways found in the hallmark curateddatabase is shown Only the pathways that are significant in at least 1 condition are shown (adjusted p value lt 025) Gray tiles are nonsignificant and theenrichment is not shown for clarity purposes (B) The boxplots show the distributions of the voom-normalized expression of claudin 18 (CLDN18) ZO-1 (TJP1)JAM-1 (F11R) and occludin (OCLN) mRNA levels using the same samples as for (A) Normalization was performed with limmavoom (C) JAM-1 proteinexpression by humanBBB ECs pretreatedwith orwithout 5μgmL antindashIL-10R2 or 5μgmL antindashIL-20R1 After 1 hour 100 ngmL rhIL-26 or control was addedfor 24 hours Samples were analyzed by Western blot (n = 3) Protein expression relative to beta-actin was calculated and normalized to the respectivecontrols (upper panels white dotted line) (D) In vitro permeability was investigated using amodified Boyden chamber assay The permeability coefficients of3-kDa 10-kDa and 70-kDa fluorescently labeled dextran through untreated (CTL) 100 ngmL IL-26 or 40 ACM (positive control) treated monolayer ofhuman BBB ECs (n = 4 experiments) Each condition was performed in triplicate (E) Transendothelial electrical resistance of human BBB ECmonolayers wasmeasured continuously for 92 hours ACM (positive control) or rhIL-26was added 24 hours after BBB EC plating (dotted line left panel and arrow right panel)The right panel is an enlarged representation of the insert box in the left panel Representative graph of n = 4 experiments is shown All conditions wereperformed in triplicate Data are represented as mean plusmn SEM (C and D) p lt 005 p lt 0001 Statistical tests Student 2-tailed t test (C) a 1-way analysis ofvariance (ANOVA) (D) and repeated-measures 1-way ANOVA followed by the Dunnett multiple comparison test (E) ACM = astrocyte-conditioned mediumBBB ECs = blood-brain barrier endothelial cells CTL = control IL = interleukin SEM = standard error of the mean


treatment significantly reduced extravasation of both IgG(figure 4B) and fibrinogen (figure 4C) in spinal cords of EAEanimals compared withHanks balanced salt solution (control)-treated mice

IL-26 therapy reduces EAE severity and limitsinfiltration of pathogenic T lymphocytesTo evaluate whether the protective effects of IL-26 on theBBB in EAE translate into a clinically detectable benefit wemonitored the symptoms of EAE mice treated with IL-26daily for 45 days We found that rhIL-26ndashtreated EAE miceexperienced a less severe clinical disease course comparedwith control-treated mice particularly during the chronicphase of the disease (figure 5 A and B) but also at the peakof the disease Surprisingly this beneficial outcome was as-sociated with an increased number of CNS-infiltratingTH lymphocytes at peak of disease and a decreased numberduring the chronic phase compared with control-treatedmice (figure 5C) A deeper analysis of these lymphocytesrevealed that the administration of IL-26 resulted in an in-filtration of both conventional and regulatory(CD4+Foxp3+CD25+) T lymphocytes (Treg) at peak ofdisease (figure e-5A linkslwwcomNXIA295 and figure5C) When analyzing cytokine production by different TH

subsets we found that the proportion and number of IL-17ndashexpressing TH lymphocytes were significantly reduced inthe CNS of rhIL-26ndashtreated animals whereas the numberand proportion of IL-10ndashexpressing TH lymphocytes wereincreased compared with control animals (figure 5D) Noclear effect was observed for IFN-γ+IL-17+ TH lymphocytesbut a tendency toward a lower proportion of GM-CSF+IL-17+ TH lymphocytes was observed (figure 5D) In the my-eloid compartment within the CNS at peak of disease nosignificant differences were found for the number of in-flammatory (Ly6C+) or patrolling (Ly6Cminus) monocytes be-tween IL-26ndashtreated and control animals (figure e-5 B andC) In addition no differences were observed for the num-bers of Ly6Cint neutrophils (figure e-5D) Of interest thenumber of microglia was decreased in the chronic phase ofIL-26ndashtreated mice compared with control mice (figuree-5E)

DiscussionAlthough IL-26 is suspected to be proinflammatory andantimicrobial15ndash171920 the exact role of this cytokine in thecontext of neuroinflammatory disorders has not been identi-fied Here we demonstrated that IL-26 in contrast to IL-17 andIL-22 produced by TH17 lymphocytes reduced BBB perme-ability through upregulation of TJ molecules thereby reducingclinical severity in EAE mice treated with rhIL-26 in vivo

Our data demonstrate that IL26 mRNA expression was in-duced in naive TH lymphocytes by a combination of theTH17-polarizing cytokines IL-1β IL-23 and TGF-β1whereas IL-6 a nonredundant cytokine for TH17 differenti-ation seems to have barely an effect on IL-26 levels In

addition IL26 transcript expression correlated strongly withTH17 markers Overall these data support its preferentialassociation with the TH17 lineage although broader regula-tion across TH lineages cannot be ruled out especially becauseour postmortem MS brain analyses show that the majority ofTH lymphocytes in perivascular cuffs express IL-26 Togetherwith our data showing nondetectable mRNA levels in B cellsmonocytes macrophages and DCs this suggests thatTH lymphocytes (especially TH17 lymphocytes) are the mostrelevant source of IL-26 in MS

Because both IL-17 and IL-22 have been shown to be in-volved in BBB disruption8 we wanted to determine whetherthe same held true for IL-26 Surprisingly IL-26 enhancedBBB integrity by reducing permeability of BBB ECs both invitro and in vivo This seemed to be a direct effect becauseboth IL-26 receptor chains were found to be expressed onBBB ECs We further demonstrated that this reduction inleakage was due to an increased expression of TJ moleculesespecially JAM-1 and CLDN5 Although our pathway anal-ysis indicated an increase in TJP1 this was not seen on theprotein level A possible explanation could be that the effecton protein levels might take longer than 24 hours Of in-terest F11R levels seemed to be decreased whereas proteinlevels were increased after 24 hours of IL-26 treatment Thiscould also be related to the time point of protein detectionHowever it is also possible that the translation rate is in-creased on IL-26 treatment Protein levels are however themost important readout for TJ molecules and hold the mostbiological relevance for BBB integrity These results incombination with the observed increase in IL-26 levels inMS suggest a compensatory mechanism to restore BBB in-tegrity and reduce BBB leakage

A barrier-promoting effect was already shown for IL-1026ndash28

which shares one of the receptor chains of the IL-26 receptorsuggesting that the barrier-promoting effect of IL-26 isestablished through signaling via the IL-10R2 subunit Wehave confirmed that both receptor units IL-10R2 and IL-20R1 are critically involved in this barrier-enhancing effectBecause it has been shown that activation of both receptorunits signals via the STAT1STAT3 pathway14 the barrier-strengthening effect of IL-26 seems to also be mediated viathis pathway Recently Itoh et al29 showed a possible op-posite effect of IL-26 on vasculature in a psoriasis model Thisdifference might either be due to a secondary inflammatoryeffect via the epidermal keratinocytes and dermal fibroblast ora direct effect To study this they used human umbilical veinECs which were able to respond to IL-26 stimulationHowever these cells did not express the IL-20R1 which wasconfirmed by the lack of p-STAT3 upregulation Our datashow that human primary BBB ECs do express the IL-20R1and that IL-26 signals via this receptor The fact that both celltypes react differently to IL-26 might not be very surprisingbecause numerous studies have already reported differenceswith regard to permeability TEER migration and chemokineproduction3031


A previous study has shown that IL-26 is not expressed in micealthough they do express a functional IL-26 receptor15 There-fore murine cell lines or mouse models have been used tofurther unravel the mechanisms of IL-26 and to study the effectof IL-26 in vivo152425 Moreover the results of these studieswere in line with findings obtained in human in vitro experi-ments To assess the role of IL-26 in vivo in the context ofneuroinflammation we administered rhIL-26 to EAE miceimmunized with MOG35ndash55 Mice treated with IL-26 displayeda milder disease course and a reduced BBB permeability Most

surprisingly there were a higher number of IL-10ndashproducingCNS-infiltrating TH lymphocytes in the IL-26ndashtreated group Inparallel we also observed a reduction in CNS-infiltratingpathogenic TH lymphocytes in IL-26ndashtreatedmice (IL-17+IFN-γ+) Whether these findings are the result of (1) suppression ofpathogenic TH lymphocytes by infiltratingTregs (2) skewing ofpathogenic TH lymphocytes toward Tregs by modification inthe cytokine environment induced by IL-26 (3) a direct effectof IL-26 on polarization of TH lymphocytes or finally (4) aneffect of IL-26 on the expression of CAMs by BBB ECs has yet

Figure 4 IL-26 treatment reduces fibrinogen and IgG leakage in EAE mice

(A) Monolayers of mouse BBB ECsimmunostained for tight junctionmolecules JAM-A (red) and claudin-5(green) and nuclei (blue) after treat-ment with IFN-γ and TNF-α (IT) 100ngmL IL-26 or left untreated (24hours treatment) Representativeimages of n = 4 experiments areshown JAM-A and Claudin-5 meanfluorescent intensity across themouse BBB EC membrane wasquantified at 30 different places perimage Three fields of view wererandomly analyzed and quantifiedfor each condition and for each ex-periment (n = 240measurements percondition) Scale bars 100 μm (B andC) MOG35ndash55 immunized C57BL6mice were injected IP with eitherHBSS or 200 ng recombinant humanIL-26 in HBSS daily from day 5 to day24 Immunohistofluorescence of IgG(green B central panel) fibrinogen(green C central panel) and PECAM-1 (red left panel) was performed onspinal cords of EAE mice at day 27postinduction Fluorescence of IgG(B right panel) and fibrinogen (Cright panel) extravasation was quan-tified (n = 3 animals per group and 3sections per mouse) Scale bars25 μm Data are represented asmean plusmn SEM (AndashC) p lt 005 p lt001 p lt 0001 Statistical tests 1-way analysis of variance followed bythe Dunnett multiple comparisontest (A) and Student 2-tailed t test (Band C) BBB ECs = blood-brain barrierendothelial cells EAE = experimentalautoimmune encephalomyelitisHBSS = Hanks balanced salt solutionIL = interleukin MOG = myelin oligo-dendrocyte glycoprotein SEM =standard error of the mean


to be investigated However it is unlikely that IL-26 directlyskews TH lymphocytes toward a regulatory phenotype becausethese cells do not express the IL-20R1 This is supported by our

data showing that rhIL-26 does not influence TH17 lymphocytepolarization (figure e-6A linkslwwcomNXIA295) In addi-tion a study by Oral et al32 also shows that IL-26 did not affect

Figure 5 IL-26 treatment ameliorates clinical symptoms of EAE

(A) MOG35ndash55 immunized C57BL6mice injected IP with either 200 μL HBSS (black squares) or 200 μL containing 200 ng recombinant human IL-26 in HBSS (grayopen circles) were scored (left panel) and weighed (normalized to day 0 right panel) daily IL-26 treatment was started at day 5 after disease induction and wasgiven daily until day 24 (B) Distribution and sumof the clinical scores for the 2 different treatment groups Each dot is 1 animal (C) The CNS of perfusedmicewascollected and infiltrating immune cells were isolated counted and analyzed by flow cytometry at onset (day 7) peak (day 15) and chronic (day 27) phases ofdisease (D) The CNS spleen and LN of perfusedmice were collected and immune cells were isolated counted and analyzed by flow cytometry for intracellularcytokine staining at peak (day 15) of disease Bars to the left of the dotted line represent cell percentages (left y-axis) and bars to the right of the dotted linerepresents total number of cells (right y-axis) Data are represented asmean plusmn SEM (AndashD) p lt 005 Statistical analyses performed Student 2-tailed t test of theareaunder the curve (A) Student 2-tailed t test (BndashD)Data shownare representative of 6 independent EAEexperimentswith9ndash30 animalsper group (AandB) n =3 animals per group (C and D) EAE = experimental autoimmune encephalomyelitis GM-CSF = granulocytemacrophage colony-stimulating factor HBSS = Hanksbalanced salt solution IL = interleukin LN = lymph nodes MOG = myelin oligodendrocyte glycoprotein SEM = standard error of the mean


naive TH lymphocyte differentiation proliferation or viabilityAn indirect effect on TH lymphocytes via antigen-presentingcells can not be excluded Recent studies show that IL-26 canbind to genomic and mitochondrial self-DNA and bacterialDNA and activate the Toll-like receptor 9 or directly activatestimulator of interferon genes in myeloid cells2033 Neverthelessthis effect of IL-26 induces IFN-α IL-6 and IL-1β which triggera proinflammatory rather than an anti-inflammatory responseAlternatively IL-26 could also act on other immune or CNScells which would in turn skew TH lymphocytes toward an IL-10ndashproducing phenotype Finally as Tregs have been reportedtomigrate faster across BBB ECs than non-Tregs34 we thereforespeculate that IL-26 produced by CNS-infiltratingTH lymphocytes in the context of neuroinflammation couldaffect the recruitment of IL-10ndashproducing TH lymphocytesacross the CNS barriers by a yet unknown mechanism Inconclusion our data reveal that IL-26 is a protective cytokinewhich is preferentially produced by TH17 lymphocytes on in-flammation During neuroinflammation the administration ofIL-26 promotes BBB functions through upregulation of TJmolecules increases the number of IL-10ndashproducing CNS-infiltrating TH lymphocytes and reduces the number of CNS-infiltrating pathogenic TH lymphocytes These findings mighthave important therapeutic implications for patients with MSwhich will be validated in future studies

AcknowledgmentThe authors are grateful to Dr D Gauchat (Flow CytometryCore Facility CRCHUM Montreal QC Canada) for hertechnical support with flow cytometry and Dr A Cleret-Buhot(Cell Imaging Core Facility CRCHUMMontreal QC Canada)for her technical support with confocal microscopy They thankDr E Haddad (Pediatric Immunologist CHU Sainte-JustineResearch Center) for providing human fetal CNS material

Study fundingThis work was funded by a grant from the Canadian Institutesof Health Research (MOP136980) A Prat holds a seniorCanada Research Chair in Multiple Sclerosis M Charabatiheld a postdoctoral studentship from the Fonds de recherchedu QuebecndashSante (FRQS) and is now supported by a doc-toral scholarship from the Multiple Sclerosis Society ofCanada (MSSC) E Peelen and S Zandee held a postdoctoralstudentship from the FRQSMSSC Partnership Award BBroux held a postdoctoral studentship from Fonds Weten-schappelijk OnderzoekndashVlaanderen L Hachehouche heldPhD studentships from the MSSC FRQS and Neuro-inflammation Training Program

DisclosureThe authors report no disclosures relevant to the manuscriptGo to NeurologyorgNN for full disclosures

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationApril 18 2020 Accepted in final form July 13 2020

Appendix Authors

Name Location Contribution

Bieke BrouxPhD

Neuroimmunology Unitand Multiple SclerosisClinic The ResearchCenter of the CentreHospitalier de lrsquoUniversitede Montreal (CRCHUM)Department ofNeuroscience Faculty ofMedicine Universite deMontreal MontrealCanada HasseltUniversity BiomedicalResearch Institute andTransnationaleUniversiteit LimburgSchool of LifeSciences DiepenbeekBelgium

Conducted experimentsanalyzed and interpreteddata and wrote themanuscript

StephanieZandee PhD

Neuroimmunology Unitand Multiple SclerosisClinic The ResearchCenter of the CentreHospitalier de lrsquoUniversitede Montreal (CRCHUM)Department ofNeuroscience Facultyof Medicine Universitede MontrealCanada

Conducted experimentsprovided importantscientific inputinterpreted the data andwrote the manuscript

ElizabethGowing MSc

Neuroimmunology Unitand Multiple SclerosisClinic The ResearchCenter of the CentreHospitalier de lrsquoUniversitede Montreal (CRCHUM)Department ofNeuroscience Faculty ofMedicine Universite deMontreal Canada

Conducted experimentsinterpreted the data andwrote the manuscript

MarcCharabati MSc



Marc-AndreLecuyer PhD


Conducted experiments

Olivier TastetMSc


Conducted experimentsand analyzed data


Appendix (continued)


LamiaHachehoucheMSc

Neuroimmunology Unitand Multiple SclerosisClinic The ResearchCenter of the CentreHospitalier de lrsquoUniversitede Montreal (CRCHUM)Department ofNeuroscience Faculty ofMedicine Universitede MontrealCanada


LyneBourbonniereMSc



Jean-PhilippeOuimet MSc



FlorentLemaitre MSc



SandraLarouche



RomainCayrol MDPhD FRCPC


Involved in the collectionof human samples andclinical characterizationof patients

AlainBouthillierMD FRCSC

Division of NeurosurgeryCentre Hospitalier delrsquoUniversite de Montreal(CHUM) Faculty ofMedicine Universite deMontreal Canada




RobertMoumdjianMD FRCSC

Division of NeurosurgeryCentre Hospitalier delrsquoUniversite de Montreal(CHUM) Faculty ofMedicine Universitede MontrealCanada


Boaz LahavBSc



Josee PoirierBSc



PierreDuquette MDFRCPC



NathalieArbour PhD


Provided isolated andpurified human fetalastrocyte cultures andprovided importantscientific input

Evelyn PeelenPhD


Conducted experimentsanalyzed and interpretedthe data and wrote themanuscript

AlexandrePrat MD PhDFRCPC


Designed the studyinterpreted the datawrote the manuscriptand secured funding


References1 Weiner HL Multiple sclerosis is an inflammatory T-cell-mediated autoimmune dis-

ease Arch Neurol 2004611613ndash16152 Nylander A Hafler DA Multiple sclerosis J Clin Invest 20121221180ndash11883 Broux B Stinissen P Hellings N Which immune cells matter The immunopatho-

genesis of multiple sclerosis Crit Rev Immunol 201333283ndash3064 International Multiple Sclerosis Genetics Consortium Beecham AH Patsopoulos

NA Xifara DK et al Analysis of immune-related loci identifies 48 new susceptibilityvariants for multiple sclerosis Nat Genet 2013451353ndash1360

5 Manel N Unutmaz D Littman DR The differentiation of human T(H)-17 cellsrequires transforming growth factor-beta and induction of the nuclear receptorRORgammat Nat Immunol 20089641ndash649

6 Hafler DA Multiple sclerosis J Clin Invest 2004113788ndash7947 Codarri L Greter M Becher B Communication between pathogenic T cells and

myeloid cells in neuroinflammatory disease Trends Immunol 201334114ndash1198 Kebir H Kreymborg K Ifergan I et al Human TH17 lymphocytes promote blood-

brain barrier disruption and central nervous system inflammation Nat Med 20071311731175

9 Croxford AL Lanzinger M Hartmann FJ et al The cytokine GM-CSF drives theinflammatory signature of CCR2 monocytes and licenses autoimmunity Immunity201543502ndash514

10 Knappe A Hor S Wittmann S Fickenscher H Induction of a novel cellular homologof interleukin-10 AK155 by transformation of T lymphocytes with herpesvirus sai-miri J Virol 2000743881ndash3887

11 Wilson NJ Boniface K Chan JR et al Development cytokine profile and function ofhuman interleukin 17-producing helper T cells Nat Immunol 20078950ndash957

12 Donnelly RP Sheikh F Dickensheets H Savan R YoungHAWalter MR Interleukin-26 an IL-10-related cytokine produced by Th17 cells Cytokine Growth Factor Rev201021393ndash401

13 Sheikh F Baurin VV Lewis-Antes A et al Cutting edge IL-26 signals through a novelreceptor complex composed of IL-20 receptor 1 and IL-10 receptor 2 J Immunol20041722006ndash2010

14 Hor S Pirzer H Dumoutier L et al The T-cell lymphokine interleukin-26 targetsepithelial cells through the interleukin-20 receptor 1 and interleukin-10 receptor 2chains J Biol Chem 200427933343ndash33351

15 Ohnuma K Hatano R Aune TM et al Regulation of pulmonary graft-versus-hostdisease by IL-26+CD26+CD4 T lymphocytes J Immunol 20151943697ndash3712

16 Dambacher J Beigel F Zitzmann K et al The role of the novel Th17 cytokine IL-26 inintestinal inflammation Gut 2009581207ndash1217

17 Corvaisier M Delneste Y Jeanvoine H et al IL-26 is overexpressed in rheumatoidarthritis and induces proinflammatory cytokine production and Th17 cell generationPLoS Biol 201210e1001395

18 Che KF Tengvall S Levanen B et al Interleukin-26 in antibacterial host defense ofhuman lungs Effects on neutrophil mobilization Am J Respir Crit Care Med 201419010221031

19 Miot C Beaumont E Duluc D et al IL-26 is overexpressed in chronically HCV-infected patients and enhances TRAIL-mediated cytotoxicity and interferon pro-duction by human NK cells Gut 2015641466ndash1475

20 Meller S Di Domizio J Voo KS et al TH17 cells promote microbial killing and innateimmune sensing of DNA via interleukin 26 Nat Immunol 201516970ndash979

21 Scala E Di Caprio R Cacciapuoti S et al A new T helper 17 cytokine in hidradenitissuppurativa antimicrobial and proinflammatory role of interleukin-26 Br J Dermatol20191811038ndash1045

22 Woetmann A Alhede M Dabelsteen S et al Interleukin-26 (IL-26) is a novel anti-microbial peptide produced by T cells in response to staphylococcal enterotoxinOncotarget 2018919481ndash19489

23 Luissint AC Artus C Glacial F Ganeshamoorthy K Couraud PO Tight junctions atthe blood brain barrier physiological architecture and disease-associated dysregula-tion Fluids Barriers CNS 2012923

24 Bao A Che KF Bozinovski S et al Recombinant human IL-26 facilitates the innateimmune response to endotoxin in the bronchoalveolar space of mice in vivo PLoSOne 201712e0188909

25 Peng YJ Wang CY Lin YH et al Interleukin 26 suppresses receptor activator ofnuclear factor kappaB ligand induced osteoclastogenesis via down-regulation of nu-clear factor of activated T-cells cytoplasmic 1 and nuclear factor kappaB activityRheumatology (Oxford) 2016552074ndash2083

26 Chen X Duan XS Xu LJ et al Interleukin-10 mediates the neuroprotection of hyperbaricoxygen therapy against traumatic brain injury in mice Neuroscience 2014266235ndash243

27 Liu Z Zhang P Ma Y et al Lactobacillus plantarum prevents the development ofcolitis in IL-10-deficient mouse by reducing the intestinal permeability Mol Biol Rep2011381353ndash1361

28 Mazzon E Puzzolo D Caputi AP Cuzzocrea S Role of IL-10 in hepatocyte tightjunction alteration inmouse model of experimental colitis MolMed 20028353ndash366

29 Itoh T Hatano R Komiya E et al Biological effects of IL-26 on T cell-mediated skininflammation including psoriasis J Invest Dermatol 2019139878ndash889

30 Man S Ubogu EEWilliams KA Tucky B CallahanMK RansohoffRM Human brainmicrovascular endothelial cells and umbilical vein endothelial cells differentially fa-cilitate leukocyte recruitment and utilize chemokines for T cell migration Clin DevImmunol 20082008384982

31 Shukaliak JA Dorovini-Zis K Expression of the beta-chemokines RANTES and MIP-1 beta by human brain microvessel endothelial cells in primary culture J NeuropatholExp Neurol 200059339ndash352

32 Oral HB Kotenko SV Yilmaz M et al Regulation of T cells and cytokines by theinterleukin-10 (IL-10)-family cytokines IL-19 IL-20 IL-22 IL-24 and IL-26 Eur JImmunol 200636380ndash388

33 Poli C Augusto JF Dauve J et al IL-26 confers proinflammatory properties toextracellular DNA J Immunol 20171983650ndash3661

34 Schneider-Hohendorf T Stenner MP Weidenfeller C et al Regulatory T cells exhibitenhanced migratory characteristics a feature impaired in patients with multiplesclerosis Eur J Immunol 2010403581ndash3590


DOI 101212NXI000000000000087020207 Neurol Neuroimmunol Neuroinflamm

Bieke Broux Stephanie Zandee Elizabeth Gowing et al function

17 lymphocytes regulates CNS barrierHInterleukin-26 preferentially produced by T

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

The abundance of immune cells and their products in CNSlesions and in the CSF of patients with multiple sclerosis (MS)together with the genetic risk conferred by immune gene vari-ants supports the concept that MS is an autoimmunedisorder1ndash5 It is strongly believed that myelin-reactive CD4+ Thelper (TH) lymphocytes are activated by an environmentaltrigger after which they cross the blood-brain barrier (BBB)invade the CNS and initiate a chronic relapsing inflammatorycascade leading to tissue damage36ndash8 These TH lymphocytesproduce cytokines such as interleukin (IL)-17 IL-22 andgranulocyte macrophage colony-stimulating factor (GM-CSF)the former 2 being involved in BBB breakdown8 and the latterstimulating CCR2+ monocytes which promote tissue damage9

IL-26 was first discovered as an inducible gene in Herpesvirussaimirindashtransformed human T lymphocytes10 but has sincebeen described as a TH17-associated cytokine that is regulatedby IL-1β IL-23 and RORγt51112 IL-26 is part of the IL-10cytokine family and binds to a heterodimeric receptor com-posed of the IL-10R2 and IL-20R1 chains which results inactivation of STAT1 and STAT31314 Although the IL26 geneis absent in rodents Ohnuma et al15 have recently demon-strated that recombinant human (rh) IL-26 can bind to andfunction through the murine receptor complex In recentyears studies regarding the role of IL-26 in various immu-nologic diseases have started to emerge15ndash20 In chronic in-flammatory diseases including Crohn disease rheumatoidarthritis chronic hepatitis C infection and chronic graft-versus-host disease IL-26 appears to play a proinflammatoryrole acting on epithelial cells monocytes natural killer cellsand fibroblasts respectively15ndash1719 It was also recently foundto be involved in antibacterial host defense1820ndash22 Howeverthe role of IL-26 inMS where TH17 lymphocytes are believedto play a major role has not yet been investigated

In the current study we hypothesized that IL-26 has aproinflammatory role and is involved in BBB disruption Wereport that the concentration of IL-26 is elevated in the serumand CSF of untreated patients with MS compared withhealthy individuals and controls respectively We furtherdemonstrate that IL-26ndashexpressing TH lymphocytes arefound within perivascular infiltrates in the brain of patientswith MS In contrast to our hypothesis we demonstrate thatthe administration of IL-26 in mice with experimental auto-immune encephalomyelitis (EAE) reduces disease severitycompared with control-treated mice This is associated with abarrier-promoting effect of IL-26 through upregulation of

tight junctionmolecules Overall our results demonstrate thatIL-26 preferentially produced by TH17 lymphocytes has abeneficial effect on barrier function during neuro-inflammation and reduces clinical and pathologic diseaseburden in sterile autoimmune CNS inflammation

MethodsEthicsBlood was obtained from patients with MS and healthy donors(HDs) after written informed consent and ethical approval(Centre Hospitalier de lrsquoUniversite de Montreal research ethiccommittee approval number BH07001) Patients were classifiedaccording to the 2010 McDonaldrsquos revised criteria for MS di-agnosis (Polman et al 2011) as previously described (Larochelleet al 2012) Other neurologic disease controls (OND) consistedof migraine glioblastoma and subjective neurologic symptomswith normal neurologic examination normal MRI and normalCSF analysis With informed consent and ethical approval(Centre Hospitalier de lrsquoUniversite de Montreal research ethiccommittee approval number BH07001) human temporal lobematerial was obtained from patients who underwent surgicaltreatment for intractable temporal lobe epilepsy and used forisolation of primary BBB endothelial cells (ECs) Human fetalbrain tissue was obtained with ethical approval (Centre Hospi-talier de lrsquoUniversite de Montreal research ethic committee ap-proval number HD07002) for isolation of astrocytes

All animal procedures were approved by the CHUM AnimalCare Committee in accordance with the guidelines of theCanadian Council on Animal Care (approval numberN15035APs)

Data availabilityData not provided in the article because of space limitationscan be shared at the request of other investigators for pur-poses of replicating procedures and results

ResultsIL-26 is preferentially expressed byTH17 lymphocytesTo address the phenotype of TH lymphocytes expressing IL-26we cultured human naive (CD45RA+) TH lymphocytes fromHDs in TH1 TH2 or TH17 differentiating conditionsWe foundthat TH17 lymphocytes expressed the highest level of IL26

GlossaryBBB = blood-brain barrier CLDN = claudins DEG = differentially expressed gene EAE = experimental autoimmuneencephalomyelitis EC = endothelial cell GM-CSF = granulocyte macrophage colony-stimulating factor GSEA = gene setenrichment analysis HDs = healthy donors IL = interleukin JAM = junctional adhesion molecule MOG = myelinoligodendrocyte glycoprotein mRNA = messenger RNA OCLN = occludin OND = other neurologic disease controls rh =recombinant humanTEER = transendothelial electrical resistance TH = T helper TJ = tight junction ZO = zonula occludens















































Appendix Authors


Bieke BrouxPhD



StephanieZandee PhD



ElizabethGowing MSc



MarcCharabati MSc






Olivier TastetMSc






LamiaHachehoucheMSc



LyneBourbonniereMSc






FlorentLemaitre MSc



SandraLarouche














Boaz LahavBSc



Josee PoirierBSc






NathalieArbour PhD



Evelyn PeelenPhD


























































Reprints

















































Appendix Authors


Bieke BrouxPhD



StephanieZandee PhD



ElizabethGowing MSc



MarcCharabati MSc






Olivier TastetMSc






LamiaHachehoucheMSc



LyneBourbonniereMSc






FlorentLemaitre MSc



SandraLarouche














Boaz LahavBSc



Josee PoirierBSc






NathalieArbour PhD



Evelyn PeelenPhD


























































Reprints






LamiaHachehoucheMSc



LyneBourbonniereMSc






FlorentLemaitre MSc



SandraLarouche














Boaz LahavBSc



Josee PoirierBSc






NathalieArbour PhD



Evelyn PeelenPhD


























































Reprints























































Reprints




interleukin-26, preferentially produced by th17 ...il-26–treated mice compared with control mice...

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