IL-11 Induces Th17 Cell Responses in Patients with Early Relapsing-Remitting Multiple Sclerosis

This information is current as Xin Zhang, Yazhong Tao, Manisha Chopra, Irena of September 26, 2021. Dujmovic-Basuroski, Jianping Jin, Yunan Tang, Jelena Drulovic and Silva Markovic-Plese J Immunol published online 20 April 2015 http://www.jimmunol.org/content/early/2015/04/17/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 20, 2015, doi:10.4049/jimmunol.1401680 The Journal of Immunology

IL-11 Induces Th17 Cell Responses in Patients with Early Relapsing-Remitting Multiple Sclerosis

Xin Zhang,* Yazhong Tao,* Manisha Chopra,* Irena Dujmovic-Basuroski,† Jianping Jin,‡ Yunan Tang,* Jelena Drulovic,† and Silva Markovic-Plese*,x

Clinically isolated syndrome (CIS) suggestive of multiple sclerosis (MS) is the earliest clinically evident phase of the disease, which may provide valuable insight into the molecular mechanisms of the initiation of the autoimmune response in MS. Our results introduce IL-11 as a new that plays a role in the autoimmune response in the early phase of the disease. IL-11 is the highest upregulated cytokine in the sera and cerebrospinal fluid from CIS patients, which is also increased in patients with clinically definitive relaps- ing-remitting MS in comparison with healthy control subjects. Serum IL-11 levels are significantly increased during clinical exacer- bations in comparison with remissions in the same patients. CD4+ cells represent a predominant cell source of IL-11 in the peripheral

circulation, and the percentage of IL-11+CD4+ cells is significantly increased in CIS patients in comparison with healthy control Downloaded from subjects. Furthermore, we have identified IL-11 as a new Th17-promoting cytokine, because it induces a differentiation of naive CD4+ T cells into Th17 cells, as well as expansion of Th17 memory cells. Because the Th17 IL-17F, IL-21 and TNF-a,andTGF-b induce differentiation of naive cells in the IL-11–secreting CD4+ cells, we propose that cross-talk between IL-11+CD4+ and Th17 cells may play a role in the inflammatory response in relapsing-remitting MS. The Journal of Immunology, 2015, 194: 000–000.

ultiple sclerosis (MS), a CNS inflammatory demye- tive RRMS, the US Food and Drug Administration has extended the http://www.jimmunol.org/ linating disease, is a leading cause of disability in the indications for immunomodulatory therapies to this early phase of M young adult, predominantly female population (1, 2). The the disease. Additional biomarkers of the early inflammatory re- autoimmune pathogenesis of the relapsing-remitting MS (RRMS) sponse are sought to more accurately identify patients amenable to has been supported by reports on the increased frequency of acti- disease-modifying therapy. vated myelin-reactive CD4+ cells in patients in comparison with The involvement of Th17 cell lineage in the development of MS healthy control subjects (HCs) (3), and the association of the myelin- has been documented by several studies (9, 10). Human Th17 cells reactive T cell expansion with the worsening of clinical disease ac- are functionally defined by the production of the effector cyto- tivity (4). However, the character of the initial disease-triggering kines IL-17A, IL-17F, IL-21, IL-22, and TNF-a (11, 12), which events remains poorly understood. Clinically isolated syndrome mediate the production of inflammatory cytokines, , by guest on September 26, 2021 (CIS) suggestive of MS is the earliest clinically evident phase of the and metalloproteinases, and promote recruitment of granulocytes disease, which may provide valuable insight into the molecular and to the sites of inflammation (13). Earlier studies mechanisms of the initiation of the autoimmune response in MS. have demonstrated an elevated IL-17A and expres- Diagnosis of CIS is established upon the first clinical presentation sion in active MS brain lesions compared with normal-appearing and magnetic resonance imaging evidence of at least two CNS de- white matter (9, 10). IL-17A is significantly in- myelinating lesions (5). Following positive results of several large, creased in the mononuclear cells derived from the blood and placebo-controlled clinical trials (6–8), in which immunomodulatory cerebrospinal fluid (csf) of MS patients, and the number of IL-17A– treatment of CIS patients postponed conversion to clinically defini- expressing blood mononuclear cells was higher during disease exacerbations than during clinically silent periods (14). Durelli et al. (15) have reported that patients with active RRMS have *Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; †Department of Neurology, Clinical Center Serbia, Belgrade 11129, about a 7-fold increase in the percentage of IL-17A–producing Serbia; ‡Center for Bioinformatics, University of North Carolina at Chapel Hill, + x CD4 cells in comparison with patients with inactive disease or Chapel Hill, NC 27599; and Department of Microbiology and Immunology, Uni- HCs, and that the number of Th17 cells in their peripheral cir- versity of North Carolina at Chapel Hill, Chapel Hill, NC 27599 culation positively correlates with the disease activity. Anti–IL- Received for publication July 3, 2014. Accepted for publication March 23, 2015. 17A mAb treatment is currently tested in a phase II clinical trial, This work was supported by National Multiple Sclerosis Society Research Grant RG 4820-A-1 (to S.M.-P.) and National Multiple Sclerosis Society Center fellowship after an initial report that this treatment induced a significant award (to Y. Tao). decrease in the number of new inflammatory CNS lesions (16). The gene expression data presented in this article have been submitted to the National Human Th17 cell differentiation is orchestrated by IL-1b, IL-6, Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi. IL-21, and IL-23, which stimulate, and IFN-g, IL-4, IL-12, IL-10, nlm.nih.gov/geo/query/acc.cgi?acc=GSE59085) under accession number GSE59085. and IL-27, which inhibit the differentiation of this cell subset, Address correspondence and reprint requests to Dr. Silva Markovic-Plese, University whereas the effect of TGF-b is still controversial (17–19). IL-6 of North Carolina at Chapel Hill, 6109 Neuroscience Research Building, 105 Mason Farm Road, Chapel Hill, NC 27599. E-mail address: [email protected] critically contributes to the expression of the Th17 transcription The online version of this article contains supplemental material. factor retinoic acid–related orphan nuclear hormone receptor Abbreviations used in this article: CIS, clinically isolated syndrome; csf, cerebrospi- (ROR)c and STAT3, and to IL-17A production in humans (19, 20). nal fluid; HC, healthy control subject; MS, multiple sclerosis; ROR, retinoic acid– Our results introduce IL-11 as a new cytokine that may play related orphan nuclear hormone receptor; RRMS, relapsing-remitting MS; SN, su- a role in the development of the autoimmune response in the early pernatant. phase of MS, because IL-11 was the highest upregulated cytokine Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 in the csf and serum derived from CIS patients in comparison with

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401680 2 IL-11 INDUCES Th17 CELL RESPONSES

HCs. Furthermore, we have identified IL-11 as a new Th17- ELISA promoting cytokine. IL-11 is a pleiotropic cytokine (21–23) and Serum and csf samples and supernatants (SNs) from the cultured CD4+ naive a member of the IL-6 family, with which it shares a gp130 signaling and memory T cells were collected and stored at 280˚C until the cytokine pathway (24). IL-11 competes with IL-6 for binding to the same measurements by ELISA. The production of IL-17A, IL-17F, IL-21, IL-22, gp130 epitopes and induces the same early response (25). IL-9 (eBioscience), IFN-g, IL-4, IL-10, TGF-b1 (BD Biosciences), and Similar to IL-6, IL-11 induces STAT3 phosphorylation in several IL-11 (R&D System) was measured following the manufacturer’s recom- mendations. For the cytokine measurements in the serum and csf samples, noninflammatory cell subsets (26–29) and induces autoreactive Ab the incubation was extended to 24 h at 4˚C, and the detection Ab incubation production by B cells (30). Several studies have reported on the IL- wasprolongedto2hatroomtemperature.Theresultsareexpressedforeach 11 anti-inflammatory effects, including the inhibition of NF-kB– subject as the cytokine concentration (in pg/ml). + mediated cytokine secretion in macrophages (31, 32) and mono- Where indicated, CD4 naive and memory T cells were isolated using a Naive and Memory CD4+ T Cell Isolation Kit (Miltenyi Biotec) from cytes (22), and the induction of apoptosis in myeloid dendritic cells fresh PBMCs derived from six CIS patients. Naive cells were differentiated (23, 33). However, in the recent literature, IL-11 has been strongly and memory cells were expanded in the presence of anti-CD3 (1 mg/ml) implicated as a proinflammatory cytokine because of its induction and anti-CD28 mAb (5 mg/ml), anti–IFN-g and anti–IL-4 mAb (10 mg/ by oxidative stress (34), its production by synoviocytes in rheu- ml), and IL-11 (10, 50, and 100 ng/ml) and anti–IL-11 (10 mg/ml) (both matoid arthritis (35), and by eosinophils in atopic dermatitis (36). from R&D Systems) for 12 d before the SN collection for the indicated cytokine measurements by ELISA. IL-11 induces bronchial inflammation (35, 36), and it is induced by In experiments that identified cytokines that induced IL-11+CD4+ dif- respiratory viral infections (37, 38). Parallel increases in the IL-11 ferentiation and expansion, T cells were isolated from six CIS patients. Naive and IL-17 gene expression have been demonstrated in the animal cells were differentiated and memory cells were expanded in the presence of model of rheumatoid arthritis (39), in inflammatory periodontal anti-CD3 and anti-CD28 mAbs, anti–IFN-g and anti–IL-4 mAbs, and indi- Downloaded from vidual cytokines previously reported to induce IL-11 secretion in other cell disease (40), and in the gastrointestinal mucosal Th17 cell inflam- types (42–46), including IL-17A, IL-17F, IL-21, IL-22, IL-23, TNF-a,TGF- matory infiltrate associated with IL-11/STAT3 overexpression (29). b1, IL-1b, IL-4, IL-6, IL-11, IL-12, and IL-13 for 12 d before the SN The chromosomal region containing the IL-11 gene (19q13) has collection. The secretion of IL-11 was measured using ELISA. been associated with susceptibility to MS (41); however, its role in The effect of IL-11 on the monocytes’ cytokine secretion was examined in the development of the inflammatory response in MS has not been three CIS patients using ELISA for the cytokine measurements in the SNs of the separated CD14+ monocytes (106 cells/condition) cultured for 48 h. elucidated. Zhang et al. (41) have reported that IL-11 expression in http://www.jimmunol.org/ MS brain lesions is primarily localized to activated astrocytes, and Affymetrix gene arrays IL-11Ra is expressed on oligodendrocytes, but the infiltrating in- Differential gene expression between the CIS patients and HCs was tested flammatory cells were not studied. In vitro studies have demon- using Affymetrix Human Gene U133 (HG-U133) arrays (Affymetrix). strated that IL-11Ra signaling increased oligodendrocyte survival PBMCs were separated from 15 CIS patients and 7 HCs, and the total RNA and proliferation via STAT3 phosphorylation (33). The same group was extracted using an RNeasy kit (Qiagen). The arrays were hybridized for has subsequently reported a significant increase in chronic progres- 16 h at 45˚C in a GeneChip Hybridization Oven 640 (Affymetrix), washed, sive experimental autoimmune encephalomyelitis activity in IL-11R and stained with R-PE streptavidin in a GeneChip Fluidics Station 400 (Affymetrix). The arrays were scanned with a Hewlett Packard GeneArray knockout mice and decreased disease activity upon administration of Scanner. Affymetrix GeneChip Microarray Suite 5.0 software was used for IL-11. However, the examined disease model is a chronic progres- washing, scanning, and basic analysis. To detect differential gene expression by guest on September 26, 2021 sive experimental autoimmune encephalomyelitis, and the study profiles between the CIS patients and HCs, we used a two-class paired test of primarily reported decreased oligodendrocyte and neuronal numbers significance analysis. Differentially expressed genes were determined using a Welch two-sample t test. A p value ,0.05 and .1.5-fold change was in the absence of IL-11 signaling, whereas the effect on the in- considered significant. flammatory response was mediated by the IL-11–inhibited prolifer- ation of CD11c+ APCs (23). RT-PCR Our study has demonstrated that the production of IL-11 is sig- CD4+ and CD8+ Tcells,CD19+ B cells, and CD14+ monocytes were sep- nificantly increased in the serum and csf samples derived from CIS arated from the PBMCs of nine CIS patients and nine age-, sex-, and race- and RRMS patients in comparison with HCs. Moreover, IL-11 serum matched HCs using CD4-, CD8-, CD19-MicroBeads (Miltenyi Biotec) and levels were significantly increased during clinical relapses of the an EasySep Negative Selection Monocyte Enrichment Kit (Stem Cell disease. CD4+ cells are the main mononuclear cell source of IL-11 Technologies). The purity of separated cells (.98%) was confirmed by FACS. The total RNA was extracted and cDNA was synthesized using a High in the peripheral circulation, and their percentage is increased in Capacity cDNA Archive Kit (Applied Biosystems). The primers for IL-1R1, CIS patients in comparison with HCs. We have demonstrated that in IL-11Ra, and 18S mRNA were purchased from Applied Biosystems, and CIS patients, IL-11 induces Th17 cell differentiation and expansion gene expression was measured by RT-PCR using TaqMan Gene Expression of Th17 memory cells, which may contribute to the autoimmune Assays (Applied Biosystems) in triplicates. The results are expressed as the response in MS. average relative gene expression and normalized against the 18S mRNA expression for each subject.

Materials and Methods Flow cytometry Study subjects For the intracellular cytokine staining, the fresh separated PBMCs from nine A total of 118 CIS patients, 59 RRMS patients, and 78 control subjects was CIS patients and nine age-, sex-, and race-matched HCs were incubated in enrolled in the study upon signing an Institutional Review Board–approved serum-free medium and stimulated with PMA (50 ng/ml) and ionomycin informed consent form. All CIS patients had experienced their first clinical (500 ng/ml) (Sigma-Aldrich) for 2 h and with BFA (1:1000 dilution; presentation consistent with demyelinating disease within a year from blood eBioscience) for an additional 3 h, as previously reported (47). The cells sample collection, had at least two magnetic resonance imaging lesions were harvested, fixed, permeabilized, and stained with FITC-conjugated consistent with MS (5), and had not received immunomodulatory therapy IL-17A (eBioscience) (when costained with IL-21, IL-22, and IL-11), IL- before the blood or csf sample collection. Control subjects that have provided 17F (R&D Systems), and IFN-g (eBioscience); PE-conjugated IL-11 csf samples had the following diagnoses: headache (8), stroke (3), mental (R&D Systems), IL-17A (when costained with all cytokines except IL- status change (3), normal pressure hydrocephalus (2), and other noninflam- 21, IL-22, and IL-11), and IL-21 (eBioscience); and allophycocyanin- matory neurologic conditions (7). Demographic data of all study subjects conjugated IL-4, IL-10, IL-9, and IL-22 (eBioscience) mAbs, as well as enrolledinthestudyarepresentedinTableI.UntreatedRRMSpatientswho PE-Cy5.5–conjugated CD4 mAb for gating. For IL-11 costaining with IL- have provided samples during the clinical attacks were enrolled in the study 21 and IL-22, cells were stained by primary Abs against human IL-11, within 30 d of the onset of the new clinical symptoms, whereas the samples in followed by FITC-conjugated secondary Abs. Isotype controls were used remission were obtained at least 30 d after the last clinical exacerbation. for determining the background. The percentages of the cells expressing The Journal of Immunology 3 each molecule were determined using a BD FACSCalibur Flow Cytometer cytokine are significantly increased during relapses compared with with CellQuest software (BD Biosciences). remissions (Fig. 1D), suggesting that IL-11 plays a role in the Fresh PBMCs derived from eight CIS patients and eight age-, sex-, inflammatory response in MS. and race-matched HCs were stained after cell permeabilization with FITC-conjugated IL-1R1, CCR6 (R&D Systems) and allophycocyanin- Gene expression profiling of the PBMCs reveals upregulation a conjugated IL-11R (Santa Cruz), IL-21R, and IL-23R (R&D Systems), as a well as PE-Cy5.5–conjugated CD4, CD8, CD19, and CD14 mAbs (BD of IL-11R in T cells from CIS patients Biosciences) and PE-conjugated IFN-g, IL-4, IL-17A, and IL-11 for gating. To capture complex gene expression changes in CIS patients, we PBMCs derived from six CIS patients were stimulated with PMA and ionomycin and stained with T-bet, GATA3, RORc, FOXP3 (eBioscience), used Affymetrix Human Gene array U133 (HG-U133) study with and p-STAT3 (BD Biosciences). 45,000 probe sets representing ∼33,000 human genes. The results of Where indicated, CD4+ naive T cells were differentiated and memory gene expression profiling in PBMCs derived from 15 CIS patients cells were expanded in the presence of anti-CD3 and anti-CD28 mAb, anti– and 7 HCs revealed 26 differentially expressed (p , 0.05, .1.5- IFN-g and anti–IL-4 mAb, and IL-11, and anti–IL-11 mAb for 12 d before fold) immune response genes (Fig. 2A), among which cytokine/ intracellular cytokine staining for RORc, T-bet, GATA3, and FOXP3 and indicated cytokines. receptors CCR1, IL-1R1, IL-11Ra, CCR6, and IL-21R In the experiments comparing the Th17-polarizing capacity of IL-11 with were significantly upregulated in CIS patients. To uncover which that of the established Th17-polarizing cytokines (IL-1b, IL-21, and IL-23), cell subsets contribute to the upregulated gene expression of these naive T cells were isolated from six CIS patients and differentiated in the cytokine and chemokine receptors, we measured their expression in presence of anti-CD3 and anti-CD28 mAb, anti–IFN-g and anti–IL-4 + + + + mAb, and IL-11 alone in the presence of Th17-polarizing cytokines, or CD4 and CD8 T cells, CD19 B cells, and CD14 monocytes. a combination of IL-11 and the Th17-polarizing cytokines for 12 d before Studies of these magnetic bead–separated cell subsets were per- the intracellular IL-17A, IFN-g, and IL-4 staining. formed in an independent cohort of nine CIS patients and nine age-, Downloaded from In the experiments that detected cytokine inducing the differentiation and + + sex- and race-matched HCs using RT-PCR. IL-11Ra was the only expansion of IL-11 CD4 cells, naive and memory T cells were isolated cytokine receptor with a significantly higher gene expression in from six CIS patients. Naive cells were differentiated, and memory cells + + were expanded in the presence of anti-CD3 (1 mg/ml) and anti-CD28 mAb CD4 and CD8 T cells in CIS patients when compared with HCs (5 mg/ml), anti–IFN-g and anti–IL-4 mAb (10 mg/ml), and individual (Fig. 2B). The increased IL-11Ra expression on CD4+ cells from cytokines including IL-17A, IL-17F, IL-21, IL-22, IL-23, TNFa, TGF-b1, CIS patients was confirmed at the protein level by the FACS IL-1b, IL-4, IL-6, IL-11, IL-12, and IL-13 (50 ng/ml) for 12 d before the staining in nine CIS patients and matched HCs (Fig. 2C). Similarly, http://www.jimmunol.org/ intracellular IL-11 staining. the gene expression of IL-11, a cytokine that binds and signals Statistics through IL-11Ra, was predominantly expressed by CD4+ Tcells + Statistical analysis of the cytokine profiling of serum and csf samples was derived from CIS patients in comparison with CD4 Tcellsfrom performed using nonparametric Mann–Whitney U test. RT-PCR, ELISA, HCs (8.3-fold; Fig. 2B, lower panel). and FACS results were analyzed using a Student paired or unpaired t test + with SigmaPlot 10.0 software (Systat Software) followed by Benjamin– Among the inflammatory cytokine-producing CD4 cells, Hochberg multiple comparison controls. Statistical analysis of the compar- IL-11+CD4+ cells are most significantly increased in CIS patients isons for multiple groups was performed using a repeated-measures ANOVA + (GraphPad Software). A p value ,0.05 was considered significant. To determine the cytokine secretion profile in CD4 cells from CIS patients, we performed intracellular cytokine staining in PBMCs by guest on September 26, 2021 from the same nine CIS patients and nine matched HCs as in Results Fig. 2C. The percentage of IL-11–producing CD4+ T cells was the IL-11 is significantly increased in the serum and csf samples most significantly increased in the CIS patients in comparison with from CIS patients in comparison with HCs the HCs. We also detected a significantly higher percentage of the Studies of the serum samples from 17 CIS patients and 28 HCs CD4+ cell–producing Th17 cytokines IL-17A, IL-17F, IL-21 and (Table I) revealed the most significantly increased levels of IL-9 IL-22, IL-9, and Th1 cytokine IFN-g–producing CD4+ cells in the (13.7-fold) and IL-11 (7.5-fold) in the CIS patients. The Th17 CIS patients. The percentages of the Th2 cytokine IL-4– and IL-10– cytokines IL-17A, IL-17F, IL-21, IL-22, and the Th1 cytokine IFN-g producing cells were significantly lower in the CD4+ Tcellsderived were also increased, whereas IL-4 levels were decreased in the from CIS patients than from their matched HCs (Fig. 3A, 3B). CIS patients (Fig. 1A). Linear correlation analysis revealed a posi- Linear correlation analysis revealed a positive correlation between tive correlation between IL-11 and IL-22 in CIS patients (r = IL-11– and IL-17F–producing cells in both CIS patients (r = 0.6881, p = 0.0023) and in HCs (r = 0.4677, p = 0.0121). Similarly, 0.8777, p = 0.0019) and HCs (r =0.6766,p = 0.0453). csf samples from 14 CIS patients and 23 control subjects with no Furthermore, intracellular IL-11 staining of the PBMCs derived evidence of CNS inflammatory disease revealed the most significant from four CIS patients revealed that CD4+ lymphocytes constitute upregulation of IL-11 (26.7-fold) in CIS patients. Th17 cytokines the predominant cell source of IL-11 in the peripheral circulation, IL-17A, IL-17F, and IL-21, the Th1 cytokine IFN-g,andTh9cy- representing 68.4% of the IL-11–producing cells. This was sig- tokine IL-9 were also increased, whereas IL-10 was significantly nificantly higher in comparison with the numbers of IL-11–pro- downregulated in comparison with the control subjects (Fig. 1B). ducing CD8+ lymphocytes, CD19+ B cells, gd T cells, and CD56+ To examine whether increased serum and csf levels of IL-11 NK cells (p , 0.001; Fig. 4A, 4B). persist in the later phase of the disease, we measured IL-11 con- We next measured the coexpression of IL-11 with T cell subsets centrations in the serum of 37 RRMS patients and in the csf samples prototypical cytokines in PBMCs from six CIS patients. Surprisingly, of 8 RRMS patients. IL-11 measurements in both compartments only a small percentage of IFN-g–, IL-4–, and IL-17–expressing showed significantly increased concentrations in comparison with cells produced IL-11 (Fig. 4C). In contrast, only a small percentage control subjects, suggesting that findings on the role of IL-11 in the of IL-11+CD4+ T cells produced IFN-g, IL-17A, IL-17F, IL-9, IL-4, early phase of the disease (CIS) are also applicable to the patients and IL-10 (Fig. 4D). Similar to the intracellular cytokine staining with clinically definitive RRMS (Fig. 1C). results, only a small percentage of IL-11+CD4+ cells expressed Th1, To demonstrate the in vivo function of IL-11 in the active phase Th2, Th17, and Treg transcription factors (Fig 4E). These results of the disease, we measured the IL-11 in the serum samples from 12 indicated that IL-11–producing CD4+ T cells might represent RRMS patients during their clinical relapses and remissions. We a distinct T cell subset, whose phenotype and function have not found that the concentration of IL-11 and the pathogenic IL-17A been previously characterized. 4 IL-11 INDUCES Th17 CELL RESPONSES

Table I. Demographic data, including age, sex, and race, for the control T cells derived from eight CIS patients expressed significantly subjects, CIS patients, and RRMS patients enrolled in this study greater levels of IL-1R1, IL-21R, and CCR6, and lower levels of IL-11R in comparison with Th1, Th2, and Th17 cells Control CIS MS (Fig. 4F). Demographic Data Subjects Patients Patients Subjects (n) 78 118 59 IL-11 induces Th17 cell differentiation Average age 6 SD (y) 41 6 13 36 6 10 41 6 13 Because IL-11Ra is expressed in a high percentage of CD4+ cells Female/male sex (n) 55/23 96/22 41/18 (76 6 12% in CIS patients; Fig. 2C), we examined the effect of IL- Race (n) + White 65 75 41 11 on naive CD4 T cell differentiation. In our preliminary study, + + + African American 13 34 14 cultures of resting naive CD45RA and memory CD45RO CD4 Asian 3 cells in the presence of IL-11 revealed significantly increased per- Native American 2 2 centages of IL-17F+CD4+ cells in naive CD4+ cells, and of IL-17A+ Unknown 4 2 cells in memory CD4+ cells, and induced production of IL-17F, IL- 21, and IL-22 in naive and IL-17A in memory cells. In contrast, IL-11 did not induce an increase in the percentage of IFN-g–and Thus, we examined the surface phenotype of IL-11+CD4+ cells, IL-4–secreting cells, nor secretion of Th1 or Th2 cytokines particularly the expression of cytokine and chemokine receptors (Supplemental Fig. 1A, 1B). Moreover, cultures of anti-CD3 + anti- that were differentially expressed in the gene expression profiling CD28 mAb–stimulated naive and memory CD4+ cells in the pres- + + + + study between CIS patients and HCs (Fig. 2A). IL-11 CD4 ence of IL-11 induced differentiation of IL-17A CD4 cells and the Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. IL-11 is significantly increased in the sera and csf from CIS patients in comparison with HCs. (A) Serum samples from 28 HCs and 17 CIS patients and (B) csf samples from 23 control subjects and 14 CIS patents were collected, and the production of indicated cytokines was measured using ELISA. Statistical analysis was performed using a nonparametric Mann–Whitney U test. Each symbol represents one subject. (C) Serum samples from 37 RRMS patients and csf from 8 RRMS patients were collected, and the production of IL-11 was measured using ELISA. Statistical analysis was performed using a repeated-measures ANOVA, *p , 0.05, ***p , 0.001. (D) Serum samples from 12 RRMS patients collected during clinical relapse (within 30 d of the onset of new symptoms) and remission (sample collected .1 mo after the last relapse) were used for IL-11 and IL-17A measurements using ELISA. Statistical analysis was performed using a paired t test. The Journal of Immunology 5 Downloaded from

FIGURE 2. Among the significantly upregulated genes for the cytokine and chemokine receptors in CIS patients, only IL-11Ra is predominantly expressed by T cells. (A) PBMCs were separated from 15 CIS patients and 7 HCs, and the total RNA was used for gene array hybridization. Tree graphs and table present 26 http://www.jimmunol.org/ immune response genes with statistically different (p , 0.05 and .1.5-fold change) expression in the CIS patients in comparison with HCs. (B)CD4+ and CD8+ T cells, CD19+ B cells, and CD14+ monocytes were separated using magnetic beads from fresh PBMCs derived from nine CIS patients and nine matched HCs. Gene expression of IL-11Ra, IL-11, and 18S mRNA was measured by RT-PCR. Results are presented as the relative gene expression, normalized against 18S mRNA. Each symbol represents one patient. Statistical analysis was performed using a repeated-measures ANOVA, *p , 0.05, ***p , 0.001. (C)FreshPBMCs derived from nine additional CIS patients and nine age-, sex-, and race-matched HCs were stained for the IL-R11a expression and gated on the CD4+ cells. Statistical analysis was performed using a paired t test. expansionofIL-17A–andIFN-g–producing memory CD4+ cells, These preliminary data set the stage for the following experiments, but not the differentiation of Th1/Th2 cells (Supplemental Fig. 1C). where we examined in greater detail a dose-titration effect of IL-11 by guest on September 26, 2021

FIGURE 3. Among CD4+ cells that produce proinflammatory cytokines, IL-11+CD4+ cells are most significantly increased in CIS patients. (A) Fresh PBMCs derived from nine CIS patients and nine matched HCs were stimulated by PMA and ionomycin before the intracellular cytokine staining. The percentage of cells expressing each cytokine was determined in gated CD4+ Tcells. Statistical analysis was performed using a paired t test with a multiple comparison correction. (B) Representative CD4+ cell intracellular cytokine staining for one CIS patient and a matched HC. 6 IL-11 INDUCES Th17 CELL RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. IL-11+CD4+ cells are predominant IL-11–producing mononuclear cells in the peripheral circulation. (A) Fresh PBMCs derived from four CIS patients were stimulated by PMA and ionomycin. The percentage of IL-11+ cells was determined in gated CD4+ and CD8+ T cells, CD19+ B cells, CD56+ NK cells, and gd TCR+ T cells. Statistical analysis was performed using a repeated-measures ANOVA. ***p , 0.001. (B) Representative staining from one CIS patient. (C) Fresh PBMCs derived from six CIS patients were stained with the indicated fluorescently-labeled Abs. The percentages of IL-11+ cells were determined in gated IFN-g–, IL-4–, and IL-17A–producing CD4+ T cells. Panels present representative staining from one CIS patient. (D) Fresh PBMCs derived from six CIS patients were stained with indicated fluorescently labeled Abs. The percentages of the cells expressing each cytokine were determined in gated IL-11+CD4+ T cells. Panels present representative staining from one CIS patient. (E) PBMCs derived from six CIS patients were stimulated with PMA and ionomycin and stained for the indicated lineage-specific transcription factors. Each symbol represents one subject. (F) Fresh PBMCs derived from eight CIS patients were stained with indicated fluorescently labeled Abs. The percentages of IL-1R1+, IL-21R+, CCR6+, and IL-11Ra+ cells were determined in gated Th1 (IFN-g+), Th2 (IL-4+), Th17 (IL-17A+), and IL-11+CD4+ T cells. Statistical analysis was performed using a repeated-measures ANOVA, *p , 0.05, **p , 0.01, ***p , 0.001. on the naive CD4 cell differentiation using the experimental ap- Naive CD4+ T cells differentiated in the presence of IL-11 proach established for the other Th17-polarizing cytokines (19). expressed RORc (Fig. 5D), the transcription factor for Th17 CD4+CD45RA+ naive T cells isolated from six CIS patients and cells, which was abrogated in the presence of anti–IL-11 mAb differentiated in the presence of anti-CD3 and anti-CD28 mAb, (Fig. 5E). In addition, the cells differentiated in the presence of anti–IFN-g and anti–IL-4 mAb, and IL-11 for 12 d showed a sig- IL-11 secreted increased levels of IL-17A, IL-17F, IL-21, IL-22, nificant dose-dependent increase in the percentage of IL-17A– and and IFN-g (Fig. 5F), whereas the secretion of IL-4, IL-9, TGF- IL-21–secreting CD4+ T cells (Fig. 5A, 5C). This effect was re- b1, and IL-10 was not induced (data not shown). Anti–IL-11 versed after blocking with anti–IL-11 mAb, but not anti–IL-6 mAb mAb abolished the induction of IL-17A, IL-17F, IL-21, and IL- (Fig. 5B). 22, but not IFN-g (Fig. 5G), suggesting that the induction of The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. IL-11 induces Th17 cell differentiation. (A) CD4+CD45RA+ naive T cells were separated from six CIS patients. A total of 2 3 106 cells per condition was stimulated by plate-immobilized anti-CD3 (1 mg/ml), anti-CD28 (5 mg/ml) mAb, anti–IFN-g and anti–IL-4 (10 mg/ml) mAbs in the absence or presence of IL-11 (10, 50, 100 ng/ml), or (B) IL-11 (50 ng/ml) plus anti–IL-11 mAbs or anti–IL-6 mAbs (10 mg/ml). After 12 d of differentiation, the cells were restimulated by PMA and ionomycin, and stained with the indicated fluorescently labeled Abs. The percentage of cells expressing IL-17A and IL-21 was determined in gated CD4+ T cells. (C) Representative staining from one experiment. (D and E) The percentage of RORc+ cells was determined in gated CD4+ T cells. (F and G) The production of the indicated cytokines was measured by ELISA in CD4+ cell SNs after 12 d of culture in the absence or presence of anti–IL-11 mAbs. Statistical analysis was performed using a repeated-measures ANOVA, *p , 0.05, **p , 0.01, ***p , 0.001. 8 IL-11 INDUCES Th17 CELL RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. IL-11 induces the expansion of Th17 memory CD4+ cells. (A and B) A total of 2 3 106 CD4+CD45RO+ T cells per condition was separated from the same six CIS patients and cultured as in Fig. 5. (C) Representative staining from one CIS patient. (D and E) The percentage of RORc+ cells was determined in gated CD4+ T cells. (F and G) The production of the indicated cytokines was measured by ELISA in CD4+ cell SNs after 12 d culture. Statistical analysis was performed using a repeated-measures ANOVA, *p , 0.05, **p , 0.01, ***p , 0.001.

IFN-g secretion is not directly mediated via IL-11, but may To determine to what extent the IL-11–induced Th17 cell dif- represent a nonspecific IFN-g induction upon anti-CD3/anti- ferentiation represents an independent pathway, or enhances the CD28 mAb stimulation. effect of the established Th17-polarizing cytokines (IL-1b, IL-6, The Journal of Immunology 9

IL-23), we induced the Th17 cell differentiation in the presence of The physiological relevance of the IL-11 concentrations measured IL-11, Th17-polarizing cytokines, or a combination of IL-11 and in the serum and csf samples from CIS patients was demonstrated by Th17-polarizing cytokines. Our results on T cells derived from six the IL-11 dose titration from 35 to 1000 pg/ml, concentrations CIS patients indicate that IL-11 increased the percentage of Th17 detected in the csf and serum samples from the CIS patients, re- cells 2.2-fold, Th17-polarizing cytokines increased it 2.5-fold, and spectively (Fig. 1). Because IL-11 at 1000 pg/ml induced naive IL-11 and the Th17-polarizing cytokines 2.9-fold, whereas those CD4+ cell differentiation into Th17 cells (Supplemental Fig. 3A, combinations of polarizing cytokines did not induce differentia- 3B), and IL-11 at both 35 and 1000 pg/ml concentrations induced tion of IFN-g and IL-4+ CD4+ cells (Supplemental Fig. 2). memory Th17 cell expansion (Supplemental Fig. 3C, 3D), we

+ confirmed the biological relevance of the ex vivo–detected serum IL-11 induces expansion of Th17 memory CD4 T cells and csf IL-11 levels. Because Th17 memory cells are predominantly involved in the To measure the effect of IL-11 on the secretion of Th-polarizing development of the autoimmune response (48), we examined the cytokines by monocytes, we measured the cytokine secretion of effect of IL-11 on the expansion of memory CD4+ cells. Studies of separated CD14+ monocytes from three CIS patients. Our results the CD45RO+ memory CD4+ cells derived from six CIS patients revealed that IL-11 induced the secretion of Th17-polarizing and expanded under the same conditions as in Fig. 5 revealed that cytokines IL-1b, TGF-b, IL-21, and IL-23 (Supplemental Fig. 4), IL-11 induces a significant dose-dependent increase in the per- although it did not change the secretion of IL-12, IL-10, and IL-27 centages of IL-17A–, IL-21–, IL-22–, and IFN-g–producing cells (data not shown).

(Fig. 6A, 6C). Anti–IL-11 mAb abolished the induction of IL-17A + + and IL-22, but not IL-21 and IFN-g, indicating that the expansion of Th17 cytokines induce differentiation of IL-11 CD4 cells Downloaded from the two latter CD4+ T cell populations was not directly induced by Because CD4+ cells represent the main source of IL-11 in the pe- IL-11 (Fig. 6B). We have identified that an increased percentage of ripheral circulation, we next examined which cytokines induce IL- memory Th17 cells expanded in the presence of IL-11–expressed 11+CD4+ cell differentiation. We induced differentiation of naive RORc (Fig. 6D), which was reversed by anti–IL-11 mAb (Fig. 6E). CD4+ T cells from six CIS patients in the absence or presence of IL- Similarly, IL-11 induced increased secretion of IL-17A, IL-17F, IL- 1b, IL-4, IL-6, IL-11, IL-12, IL-13, IL-17A, IL-17F, IL-21, IL-22,

21, IL-22, and IL-9 by memory cells (Fig. 6F), which was reversed IL-23, TGF-b1, and TNF-a, cytokines reported to induce IL-11 http://www.jimmunol.org/ for IL-17A, IL-17F, and IL-21 by anti–IL-11 mAb (Fig. 6G). In secretion in the other cell types (42–45). Our data indicate that contrast, IL-11 failed to increase the expression of T-bet, GATA3, or Th17 cytokines IL-17F, IL-21, TNF-a,aswellasTGF-b1 signifi- FOXP3, or the secretion of IFN-g,IL-4,TGF-b1, or IL-10 in cantly increased the percentage of IL-11+CD4+ T cells after 12 d of memory CD4+ cells (data not shown). differentiation (Fig. 7A). In addition, IL-1b and TGF-b1alsosig- by guest on September 26, 2021

FIGURE 7. Th17 cytokines induce differentia- tion of IL-11–producing cells. (A)CD4+CD45RA+ naive T cells were separated from six CIS patients. Atotalof23 106 cells per condition was stimu- lated by plate-immobilized anti-CD3 (1 mg/ml), anti-CD28 (5 mg/ml) mAb, anti–IFN-g, and anti– IL-4 (10 mg/ml) mAbs in the absence or presence of the indicated cytokines (50 ng/ml). After 12 d of differentiation, the cells were restimulated by PMA and ionomycin, and stained with the fluorescein- labeled Abs against IL-11 and CD4. The percentage of cells expressing IL-11 was determined in gated CD4+ T cells. Statistical analysis was performed using a paired t test. (B)TheconcentrationofIL-11 in the SNs from the above cultures was measured using ELISA. (C)CD4+CD45RO+ memory T cells were separated from six CIS patients, and 2 3 106 cells per condition were cultured as indicated in (A). After 12 d, the SNs were collected, and the IL-11 concentration was measured by ELISA. 10 IL-11 INDUCES Th17 CELL RESPONSES nificantly induced IL-11 secretion by the naive CD4+ Tcellsduring IL-17F, IL-21, and IL-22 cytokine secretion. Although IL-11–in- differentiation (Fig. 7B). duced Th17 differentiation was inhibitedbyanti–IL-11mAb,itwas To characterize the expansion of IL-11+CD4+ memory cells, we not inhibited by anti–IL-6 mAb, indicating that IL-11–induced Th17 activated memory CD4+ cells from the same six CIS patients in cell differentiation is independent of IL-6. Although IL-11 alone can the absence or presence of the same cytokines as in Fig. 7A. We induce Th17 cell differentiation, our experiments revealed that the found that none of the tested cytokines induced the expansion of combination of IL-11 and the Th17-polarizing cytokines (IL-1b, IL-11+CD4+ T memory cells (data not shown). However, IL-17F, IL-6, and IL-23) most effectively induced Th17 cell differenti- TNF-a, IL-1b, TGF-b1, and IL-11 increased the production of IL- ation. Therefore, IL-11 induces an independent, but also a com- 11 by memory CD4+ T cells (Fig. 7C). plementary pathway to the other Th17-polarizing cytokines. Given significantly higher numbers of memory Th17 cells (48) Discussion and their capacity to produce high amounts of cytokines in com- Cytokine profiling of serum and csf samples obtained at the time of parison with the naive cells, we examined the role of IL-11 in the CIS diagnosis revealed that IL-11 was the most significantly in- expansion of memory Th17 cells in CIS patients. Our results creased cytokine in comparison with control subjects, which suggests revealed that the activation of CD45RO+ memory CD4+ cells in the the potential involvement of IL-11 in the autoimmune response in presence of IL-11 induced expansion of the IL-17A–, IL-21–, and early MS. Similar findings were documented in patients with clin- IL-22–producing RORc+ Th17 cells. ically definitive RRMS, suggesting the proinflammatory role of Whereas the average IL-11 concentration detected in the sera of IL-11, which was further confirmed by its significantly higher CIS patients induced differentiation of naive Th17 cells and the serum concentrations during the clinical relapses in comparison expansion of memory Th17 cells, the IL-11 concentration detected Downloaded from with the remissions of untreated RRMS patients. in the csf was only able to induce the expansion of memory Th17 An independent Affymetrix gene array study of PBMCs derived cells. A prominent response of memory Th17 cells to the low from CIS patients has identified an increased expression of several concentrations of IL-11 may play a role in the amplification of Th17- cytokine and chemokine receptors, which may reflect patients’ mediated autoimmune response in the CNS of CIS patients. constitutive susceptibility for an enhanced inflammatory response. Regarding the regulation of IL-11 secretion, previous studies + Our results revealed that a high percentage of CD4 T cells express have reported that the IL-11 secretion was induced by IL-17F in http://www.jimmunol.org/ IL-11Ra (44 6 29% in HCs and 76 6 22% in CIS patients), bronchial epithelial cells (42), by IL-22 in myofibroblasts (46), by whereas only a low percentage of CD4+ T cells secrete IL-11 (1.3 6 IL-1b in fibroblast and stromal cells (43, 44), and by 0.4% in HCs and 2.8 6 1.1% in CIS patients). Those results are TGF-b in fibroblast (43). In this study, we found that Th17 cyto- reflected in the gene array results from the bulk PBMCs. Confir- kines IL-17F, IL-21, and TNF-a, as well as TGF-b1, induced the matory RT-PCR studies of the IL-11Ra expression in multiple cell differentiation of IL-11+CD4+ T cells. Interestingly, although none subsets have identified its predominant expression in T cells, which of the tested cytokines induced the expansion of memory IL-11+ was increased in CIS patients, accompanied by a similar expression CD4+ Tcells,IL-17F,TNF-a, TGF-b1, IL-1b, and IL-11 induced pattern of its binding cytokine IL-11. CD4+ lymphocytes constitute IL-11 secretion by memory CD4+ T cells. The induction of IL-11+ the main cellular source of IL-11 in the peripheral circulation, CD4+ T cell differentiation and IL-11 secretion in response to the by guest on September 26, 2021 representing 68.4% of the IL-11–producing PBMCs. Whereas the above early proinflammatory cytokines may explain an increased previous studies identified stromal cells, including synoviocytes IL-11 level in serum and csf samples from CIS patients. In addition (35), lung fibroblasts, epithelial cells (49), and eosinophils (36), as to the IL-11–induced Th17 cell differentiation, the induction of IL- a source of IL-11, our findings demonstrated that IL-11 is also 11+CD4+ T cell differentiation by Th17 cytokines inferred a posi- produced by multiple peripheral blood mononuclear, predominately tive feedback mechanism by which Th17 cells cross-talk with IL- CD4+ T cells. Furthermore, we identified a subset of CD4+ Tcells 11+CD4+ cells and may amplify the inflammatory response in the that secrete IL-11, justifying their more detailed study in the early stage of MS. pathogenesis of MS. The surface marker phenotype of IL-11+CD4+ T cells was Acknowledgments characterized by significantly higher IL-1R1, IL-21R, and CCR6 We thank Dr. Rick Meeker, Diane Armao, and Kevin Blauth for critical + and lower IL-11Ra expression in comparison with the IFN-g reading of the manuscript. Affymetrix gene array hybridization was per- (Th1), IL-4+ (Th2), and IL-17A+ (Th17) cells. Increased expression formed at the University of North Carolina Neuroscience Center gene ex- of IL-1R1 on IL-11+CD4+ T cells is consistent with the reported pression profiling core facility. role of IL-1 in the induction of IL-11 secretion in fibroblasts (43, 45) and in CD4+ cells, as found in our study (Fig. 7B, 7C). IL-21 is Disclosures an autocrine cytokine secreted by Th17 cells, whose binding to IL- The authors have no financial conflicts of interest. 21R promotes or maintains Th17 lineage commitment (50, 51) and + + also induces the differentiation of IL-11 CD4 naive cells (Fig. 7A). 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