IL-11 Induces Encephalitogenic Th17 Cells in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis

This information is current as Xin Zhang, Nazanin Kiapour, Sahil Kapoor, Tabish Khan, of September 27, 2021. Madhan Thamilarasan, Yazhong Tao, Stephanie Cohen, Ryan Miller, Raymond A. Sobel and Silva Markovic-Plese J Immunol published online 24 July 2019 http://www.jimmunol.org/content/early/2019/07/23/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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 24, 2019, doi:10.4049/jimmunol.1900311 The Journal of Immunology

IL-11 Induces Encephalitogenic Th17 Cells in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis

Xin Zhang,* Nazanin Kiapour,* Sahil Kapoor,* Tabish Khan,* Madhan Thamilarasan,* Yazhong Tao,* Stephanie Cohen,† Ryan Miller,‡ Raymond A. Sobel,x and Silva Markovic-Plese*,{,‖

IL-11+CD4+ cells accumulate in the cerebrospinal fluid of patients with early relapsing-remitting multiple sclerosis (MS) and in active brain MS lesions. Mouse studies have confirmed a causal role of IL-11 in the exacerbation of relapsing-remitting exper- imental autoimmune encephalomyelitis (RREAE). Administration of IL-11 at the time of clinical onset of RREAE induced an acute exacerbation and increased clinical scores, which persisted during the entire course of the disease. IL-11 increased the numbers of spinal cord inflammatory foci, as well as the numbers of peripheral and CNS-infiltrating IL-17+CD4+ cells and IL-17A serum levels. Ag recall assays revealed that IL-11 induces IL-17A+, GM-CSF+, and IL-21+CD4+ myelin Ag-reactive cells. Passive Downloaded from transfer of these encephalitogenic CD4+ T cells induced severe RREAE with IL-17A+CCR6+ CD4+ and B cell accumulation within the CNS. Furthermore, passive transfer of nonmanipulated CNS-derived mononuclear cells from mice with RREAE after a single dose of IL-11 induced severe RREAE with increased accumulation of IL-17A+ and CCR6+ CD4+ cells within the CNS. These results suggest that IL-11 might serve as a biomarker of early autoimmune response and a selective therapeutic target for patients with early relapsing-remitting MS. The Journal of Immunology, 2019, 203: 000–000. http://www.jimmunol.org/

mmunomodulatory therapies are most effective when ad- cerebrospinal fluid (CSF) and serum in comparison with healthy ministered early in the course of relapsing-remitting multiple control (HC) subjects. Moreover, IL-11 serum levels were sig- I sclerosis (RRMS). Therefore, we have been searching for nificantly higher in relapses than in the remissions of untreated biomarkers of the early autoimmune response to accurately RRMS patients, suggesting the involvement of this in the identify patients with clinically isolated syndrome (CIS) suggestive pathogenesis of RRMS. In vitro studies have revealed that IL-11 of multiple sclerosis (MS), who are amenable to early disease- induces Th17 cell differentiation and expansion in CIS patients modifying therapies (1). Our previous study in CIS patients has (2). Our human studies have identified that CD4+ cells represent a identified IL-11 as the most significantly increased cytokine in the predominant source of IL-11 within the peripheral circulation. In by guest on September 27, 2021 comparison with healthy donors, IL-11+CD4+ cells from CIS patients were significantly increased in the peripheral circulation *Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, + NC 27599; †Lineberger Cancer Institute, University of North Carolina at Chapel Hill, and exhibited the highest CCR6 expression (86%) among CD4 Chapel Hill, NC 27599; ‡Department of Pathology, University of North Carolina at T cell subsets, which implied their potential for early migration to Chapel Hill, Chapel Hill, NC 27599; xDepartment of Pathology, Stanford University, { the CNS (3). Palo Alto, CA 94394; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; and ‖Department of Neurol- IL-11 is a member of the IL-6 cytokine family, whose proto- ogy, Thomas Jefferson University, Philadelphia, PA 19107 typical cytokine promotes Th17 differentiation in both mice and ORCIDs: 0000-0002-7473-415X (N.K.); 0000-0003-3553-382X (S.K.); 0000-0002- humans (4). However, IL-6 alone does not induce Th17 differ- 0096-8762 (R.M.); 0000-0002-0477-9002 (R.A.S.). entiation, in contrast to IL-11, which induces Th17 cell differen- Received for publication March 14, 2019. Accepted for publication July 1, 2019. tiation and expansion that were selectively blocked by aIL-11 This work was supported by National Multiple Sclerosis Society Research Grant G mAb and not by aIL-6 mAb (2). These previously reported human 4820-A-1; National Institutes of Health Grants R03 AI111592, R01 AI 131238, and R01 AI 131238; and University of North Carolina Translational and Clinical Sciences in vitro studies prompted current in vivo studies of the causative Pilot Award 550KR61329. role of IL-11 in the development of the Th17 autoimmune responses X.Z. designed and performed experiments and wrote the manuscript; N.K., S.K., T.K., in relapsing-remitting experimental autoimmune encephalomyelitis M.T., and Y.T. performed experiments; S.C. quantified human immunohistochemistry (RREAE), an animal model of RRMS. studies; R.M. analyzed MS lesion pathology; R.A.S. analyzed mice pathology; and S.M.-P. designed the study and wrote the manuscript. In the presence of the ligand-binding subunits IL-6Ra and IL-11Ra, IL-6 and IL-11 bind to the signal transduction unit Address correspondence and reprint requests to Dr. Silva Markovic-Plese, Depart- ment of Neurology, Thomas Jefferson University, 900 Walnut Street, Philadelphia, gp130 at overlapping epitopes, leading to the formation of ternary PA 19107. E-mail address: [email protected] complexes with similar downstream signaling (5). STAT3, a tran- The online version of this article contains supplemental material. scription factor involved in Th17 differentiation (6), is activated in Abbreviations used in this article: BBB, blood–brain barrier; CIS, clinically isolated response to IL-6/IL-6R and IL-11/IL-11R signaling (5). Our labo- syndrome; CSF, cerebrospinal fluid; EAE, experimental autoimmune encephalomy- ratory and others have reported that IL-11Ra is expressed by mul- elitis; EC, endothelial cell; HC, healthy control; LN, lymph node; MRI, magnetic resonance imaging; MS, multiple sclerosis; PLP, proteolipid ; RR, relapsing- tiple PBMC subsets, with predominant expression in T cells (2, 7). remitting; RREAE, relapsing-remitting experimental autoimmune encephalomyelitis; Zhang et al. (8) reported that IL-11 expression in chronic brain RRMS, relapsing-remitting multiple sclerosis. MS lesions is primarily localized to activated astrocytes at the This article is distributed under The American Association of Immunologists, Inc., lesion border, whereas IL-11Ra is expressed on oligodendrocytes. Reuse Terms and Conditions for Author Choice articles. However, the inflammatory cells within lesions were not studied. Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 In vitro studies conducted by the same group have demonstrated

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900311 2 IL-11 INDUCES ENCEPHALITOGENIC Th17 CELLS that IL-11Ra signaling increases oligodendrocyte survival and peripheral lymphoid tissues. Spinal cord and brain tissue were cut into proliferation via STAT3 phosphorylation (9). Although we ac- small pieces and digested in PBS containing Collagenase D (5 mg/ml) knowledge the reported findings on the role of IL-11 in chronic (Roche) for 45 min at 37˚C, with a short vortex every 15 min. After digestion, the cells were passed through a cell strainer and washed MS that suggest increased in vitro oligodendrocyte differentiation with PBS, followed by 38% Percoll gradient for separation of the CNS and survival, we propose that the proinflammatory effect of IL-11 mononuclear cells. may prevail in the context of active MS lesions and the expression of IL-11R on T cells, monocytes (1), and B cells (10), which Flow cytometry (FACS) exhibit an inflammatory response to IL-11. Cells were separated from the blood and CSF samples of RRMS patients or In the current study, we found that IL-11+CD4+ cells are sig- from the blood and tissues of mice with RREAE that received a single nificantly enriched in the CSF of RRMS patients in comparison injection of IL-11 or control vehicle as well as from the recipients of the LN CD4+ cells or CNS-derived mononuclear cells. The cells were stimulated with their matched blood samples. Immunohistochemistry studies with PMA (50 ng/ml) and ionomycin (500 ng/ml) (Sigma-Aldrich) for 2 h of active brain MS lesion biopsy samples revealed an enrichment and with BFA (1:1000 dilution) (eBioscience) for an additional 3 h for the of IL-11+CD4+ cells in comparison with the peripheral circulation, intracellular staining, as previously reported (2, 12). The cells were har- suggesting a role for this cytokine in the development of inflam- vested, fixed, permeabilized, and stained with fluorescently conjugated matory CNS lesions. Animal studies have confirmed the causal Abs against human IL-11 (R&D Systems), IL-17A, IFN-g, (eBioscience), and CD4 (BD Bioscience) or murine IL-17A, IFN-g, IL-4, TNF-a, IL-1b, role of IL-11 in the induction of acute exacerbations and increased IL-10, CD11b, CD8a, CD19 (eBioscience), Ly6C, CCR6, ICAM-1, VLA-4 clinical scores throughout the course of RREAE. A single dose and CD4 (BD Bioscience), GM-CSF (Miltenyi Biotec), IL-21, IL-21R, and of IL-11 significantly increased the number of CNS-infiltrating IL-6R (R&D Systems). Isotype controls were used to determine the

IL-17A+CD4+ cells and IL-17A serum levels in comparison with background. The percentage of cells expressing each molecule was de- Downloaded from termined in gated cells using a BD FACSCalibur Flow Cytometer with control RREAE mice. Ag recall experiments with spleen and CellQuest software (BD Biosciences). lymph node (LN) cells from proteolipid protein (PLP)139–151- immunized mice revealed that IL-11 induces encephalitogenic Immunohistochemistry + CD4 cells, characterized by IL-17A, GM-CSF, and IL-21 se- Brain MS lesion tissue was obtained from five patients using a stereotactic cretion, which, upon passive transfer, induce severe RREAE needle biopsy. Sequential dual immunofluorescent staining of brain sections + + + with IL-17A CCR6 CD4 cell and B cell accumulation within was performed in a Bond fully automated slide staining system (Leica http://www.jimmunol.org/ the recipients’ CNS. Furthermore, passive transfer of CNS- Biosystems) using Bond Research Detection (DS9455) and Bond Polymer derived, nonmanipulated mononuclear cells from mice with ac- Refine Detection (DS9800) kits. Slides were deparaffinized in Bond dewax solution (AR9222) and hydrated in Bond wash solution (AR9590). Mouse tive RREAE following a single IL-11 administration induced mAb against human CD4 (clone 4B12, NCL-L-CD4-368) was from Leica more-severe disease, with increased accumulation of IL-17A+ Biosystems (Norwell, MA). Rabbit polyclonal Abs against human IL-11 and CCR6+ CD4+ cells within the recipient CNS, in comparison (sc-7924) and IL-17A (HPA052258) were from Santa Cruz Biotechnol- with the passive transfer of nonmanipulated CNS-derived cells ogy (Dallas, TX) and Sigma-Aldrich (St. Louis, MO), respectively. IL-11 epitope retrieval was done for 30 min at 100˚C in Bond epitope retrieval from RREAE mice. solution 1 (pH 6) (AR9661) and in solution 2 for IL-17A (at pH 9) (AR9640). After pretreatment, slides were incubated for 1 h with anti–IL-11 Materials and Methods (1:50) or anti–IL-17A (1:200) mAb followed with Leica goat anti-rabbit by guest on September 27, 2021 polymer DS (RE72290-K) and the tyramide Cy5 (no. SAT705A001EA; Study subjects PerkinElmer, Boston, MA). The unmasking of CD4 epitope was done for Six RRMS patients were enrolled in the study upon signing an Institutional 10 min in Bond epitope retrieval solution 2. Anti-CD4 mAb (1:200) was Review Board–approved consent form. The inclusion criteria for patients applied for 30 min, followed with the secondary Ab from a Bond Polymer consisted of an RRMS diagnosis (11) and age of 27–55 (average 43.3 6 Refine kit (DS9800) and tyramide Cy3 (SAT704A001EA; Perkin Elmer). 10.6); all patients were female, with five being white, and one being Nuclei were stained with Hoechst 33258 (no. H3569; Life Technologies, African American. The exclusion criterion was prior treatment with Grand Island, NY). The stained slides were mounted with ProLong Gold immunomodulatory or immunosuppressive therapy. Brain biopsy Antifade Reagent (no. P36934; Life Technologies). Digital imaging and 3 samples were obtained for diagnostic purposes from an additional five analysis were performed using high-resolution acquisition ( 20 objective) untreated RRMS patients, age 20–66 (average 39.6 6 17.3), under a of the stained slides in the DAPI, Cy3, and Cy5 channels using the Aperio protocol approved by the UNC Institutional Review Board. Supplemental ScanScope FL (Leica Biosystems). Cell nuclei were visualized in DAPI Table I presents brain magnetic resonance imaging (MRI) and pathology channel (blue), CD4 were visualized in Cy3 channel (green), and IL-11 findings from patients that underwent diagnostic brain lesion biopsy. and IL-17A were visualized in Cy5 (red). For all slides, demyelinated regions on images were annotated by a neuropathologist based on H&E Active experimental autoimmune encephalomyelitis and Luxol fast blue staining. Automated digital analysis of the images was run in both myelinated and demyelinated regions. Tissue Studio software All animal procedures were approved by the Institutional Animal Care and (Tissue Studio version 2.4 with Tissue Studio Library version 4.0; Definiens, Use Committee of the University of North Carolina at Chapel Hill. Eight- to Carlsbad, CA) was used to enumerate cells that coexpress the biomarkers of twelve-week-old female SJL/J mice purchased from Charles River Labo- interest in the annotated regions. ratories were sacrificed as HCs or immunized with 80 mg of PLP139–151 For the mouse study, on day 70 postimmunization, six mice per peptide per mouse in CFA containing Mycobacterium tuberculosis (4 mg/ml). group that received IL-11 or control vehicle were sacrificed, and on day Pertussis toxin (200 ng) was injected i.p. on day 0 and day 2, following im- 70 post–passive transfer, six mice per group that received IL-11– munization. Starting from day 12 postimmunization, 12 mice per group were polarized CD4+ or control Ag-specific CD4+ cells were sacrificed, and the i.p. injected daily with either recombinant mouse IL-11 (0.5 mg/mouse) brain and spinal cords were removed and fixed in 4% paraformaldehyde. (Peprotech) or control vehicle (PBS) for 10 d. Clinical scores were assigned Paraffin sections were stained with Luxol fast blue and H&E. Inflammatory daily as follows: 1) limp tail, 2) hind limb weakness, 3) hind limb paralysis, 4) infiltrates were quantified as described in the previous study by a blinded hind limb paralysis and forelimb weakness, 5) moribund mice, and 6) death. neuropathologist (13). The mice were monitored for 70 d for the clinical scores, followed by histo- logical studies. Western blotting Where indicated, on day 12 and on day 56 postimmunization, six mice per group were i.p. injected with recombinant mouse IL-11 (0.5 mg/mouse) In an in vitro signaling experiment, naive mice spleen cells were incubated or control vehicle (PBS). After 16 h, the mice were sacrificed. Blood was in the absence or presence of IL-11 (R&D Systems) dose titration and collected via cardiac puncture, and the mice were perfused with 50 ml of aIL-11 mAb at 10 mg/ml (R&D Systems). Total and phosphorylated PBS containing heparin (10 U/ml) (Sigma-Aldrich). LNs, including LN1 STAT3 (), RORgt,andb actin expression was determined (superficial cervical, deep cervical, axillary, and brachial LNs) and LN2 after 30 min. (inguinal LNs); spleen; spinal cord; and brain tissue were harvested. Sera Cell lysates from the spleen cells derived from four mice with RREAE were collected for ELISA, and cells were separated from blood and and four mice with RREAE that received a single dose of IL-11 were The Journal of Immunology 3

FIGURE 1. IL-11+CD4+ andTh17cellsareenrichedintheCSFfromRRMSpatientsincomparisonwiththematchedbloodsamples.(A) PBMCs and CSF cells were separated from blood and CSF samples from six untreated RRMS patients. The cells were stimulated with PMA and ionomycin for intracellular staining. The percentage of cells expressing the indicated was determined in gated CD4+ T cells. Each symbol represents an individual donor, and horizontal bars represent mean value. Statistical analysis was performed using a two-tailed paired Student t test. (B) Representative staining of the indicated cytokines in the gated CD4+ cells from one out of six untreated RRMS patients.

denatured in SDS and resolved by 10–15% SDS-PAGE depending on the expressed for each mouse as the cytokine concentration in picogram per Downloaded from m.w. of the protein of interest. The were electroblotted onto PVDF milliliter. membranes (Bio-Rad). The membranes were probed with primary Abs against mouse NF-kB p65 (Cell Signaling), RORgt (R&D Systems), Ag recall assay and b-actin (Santa Cruz Biotechnology), followed by HRP-conjugated IgG secondary Ab (Santa Cruz Biotechnology) incubation. Protein Eight- to ten-week-old female SJL/J mice with active experimental phosphatase 2A inhibitor (Santa Cruz Biotechnology) was added to the autoimmune encephalomyelitis (EAE) were sacrificed at day 11 sample. The protein expression was detected by Imagequant LAS 4000 postimmunization. Spleen and LN cells were harvested and cultured in software (GE Healthcare Life Sciences). Quantification of band den- the absence or presence of PLP139–151 (20 mg/ml), IL-11 (100 ng/ml), http://www.jimmunol.org/ sities was performed using Image Quant TL software (GE Healthcare and IL-23 (20 ng/ml) for 7 d. The percentage of cells expressing the Life Sciences). indicated intracellular cytokines and surface markers was determined in gated CD4+,CD8+,andCD19+ cells by flow cytometry. RT-PCR Passive transfer RREAE Cells were separated from the blood and tissues of RREAE mice that re- ceived a single dose of IL-11 or control vehicle. The total RNA was LN cells were separated from donor RREAE mice at day 11 postimmunization extracted, and cDNAwas synthesized using a High-Capacity cDNA Archive and cultured in three conditions: 1) PLP139–151 (20 mg/ml), 2) PLP139–151 + Kit (Applied Biosystems). The primers for IL-17A, IFN-g, IL-4, IL-21, IL-22, IL-11 (100 ng/ml), or 3) PLP139–151 + IL-23 (20 ng/ml) for 7 d. After 7 d IL-11, RORc, T-bet, GATA3, AHR, and 18S mRNA were purchased from culture, CD4+ T cells were separated using a CD4+ magnetic bead 6 + Applied Biosystems, and expression was measured by RT-PCR using isolation kit (Miltenyi Biotech). A total of 20 3 10 CD4 Tcells by guest on September 27, 2021 TaqMan Assays (Applied Biosystems) in triplicate. The re- (purity .98%) per recipient were resuspended in 500 ml13 PBS for sults are expressed as the average relative gene expression and normalized passive transfer via i.p. injection. Clinical scores were assigned daily for against the 18S mRNA expression. 70 d in recipient mice. ELISA Statistical analysis Serum samples were collected from mice with RREAE that received a The results of the clinical scores, FACS, RT-PCR, ELISA, and histology single dose of IL-11 or control vehicle. The production of IL-11 (R&D studies are presented as means and, where indicated, SD. They were an- Systems) and IL-17A (eBioscience) was measured, as previously reported alyzed using a two-tailed Student paired or unpaired t test. The comparison (2). The sample incubation was extended to 24 h at 4˚C, and the detection of multiple groups was performed using a repeated measures ANOVA Ab incubation was prolonged to 2 h at room temperature. The results are (GraphPad Software).

FIGURE 2. Immunohistochemistry studies of the active brain MS lesions reveal accumulation of IL-11+CD4+ cells in the inflammatory infiltrates. (A) Active brain MS lesion tissue from five patients obtained by biopsy was used for CD4, IL-11, and IL-17A staining. Presented is an average percentage of IL-11+ and IL-17A+ CD4+ cells 6 SD in the myelinated and demyelinated tissue. (B) Representative staining from one out of five patients. Cell nuclei were visualized in DAPI channel (blue), CD4 in Cy3 channel (green), and IL-11 and IL-17A in Cy5 (red). Scale bars, 1 mm (H&E and LFB panels). 4 IL-11 INDUCES ENCEPHALITOGENIC Th17 CELLS

FIGURE 3. IL-11 induces STAT3 phosphorylation and RORgt expression. (A) Spleen cells from nine naive SJL/J mice were cultured in the absence or presence of IL-11 (100 ng/ml), followed by intracellular staining for IL-17A and surface staining for CCR6. The percentage of cells expressing each molecule was determined in gated CD4+ T cells. Each symbol represents one mouse; horizontal bars represent mean values. Statistical analysis was performed using a two-tailed paired Student t test. (B) Spleen cells were separated from three naive SJL/J mice and incubated in the absence or presence of IL-11 (10, 50, 100 ng/ml) and aIL-11 mAb. Cell lysates were prepared following 30 min of culture and used for Western blotting to detect total and pSTAT3, STAT3, RORgt, and b-actin expression. Downloaded from

Results Immunohistochemistry studies of diagnostic brain MS IL-11+CD4+ and Th17 cells are enriched in the CSF and brain lesion biopsy samples (from five additional patients whose lesions of RRMS patients demographic, brain MRI, and pathology data are presented in the Supplemental Table I) revealed an enrichment of IL-11+CD4+ Our studies revealed that serum IL-11 levels correlate with the cells within the infiltrating cells in both myelinated (average 47.9%)

concomitant brain MRI T2 and T1 lesion volumes (X. Zhang, http://www.jimmunol.org/ and demyelinated areas (average 62.8%) (Fig. 2) in comparison unpublished results), supporting the role of this cytokine in CNS with the percentages of IL-11+CD4+ cells in peripheral circulation lesion formation (14). (Fig. 1A). The results suggest an early accumulation of IL-11+CD4+ We have reported that the percentage of IL-11+CD4+ cells in the cells in the active MS lesions. peripheral blood is significantly increased in CIS patients, in comparison with the age-, sex-, and race-matched HCs (2). Those IL-11 induces the expansion of IL-17A– and CCR6-expressing + cells highly express CCR6, implying their potential for the mi- CD4 T cells gration to the CNS via a similar mechanism as Th17 cells (3). To examine the causative effect of IL-11 in the induction of Indeed, the flow cytometry studies of cells derived from the CSF Th17 cell responses and on their migration to the CNS, we have and concomitant blood samples from the six recently diagnosed, extended our studies to RREAE in SJL/J mice, an established animal by guest on September 27, 2021 untreated RRMS patients (average age 43.3 6 10.6 y, all female, model of the disease. Studies of the spleen cells from nine naive SJL/J five white, one African American) revealed that the mean per- mice revealed that IL-11 in vitro stimulation induces a significant centage of IL-11+CD4+, IL-17A+CD4+, and IL-11+ IL-17A+CD4+ expansion of IL-17A+CD4+ and CCR6+CD4+ cells (Fig. 3A). IL-11 T cells were significantly increased in the CSF in comparison with induced STAT3 phosphorylation and RORgt expression in a dose- the matched PBMCs, indicating the accumulation of those cells in dependent fashion, which was reversed in the presence of aIL-11 the CSF (Fig. 1). mAb (Fig. 3B).

FIGURE 4. IL-11 induces worsening of the clinical disease course in RREAE. (A) Twelve fe- male 8- to 12-wk-old SJL/J mice were immunized with 80 mg/mouse of PLP139–151 peptide. Starting from day 12 postimmunization, six mice per group received i.p. injections of recombinant mouse IL-11 (0.5 mg/mouse) or control vehicle daily for 10 d. Clinical scores were assigned daily for 70 d. Presented are mean clinical scores 6 SD. Statistical analysis was performed using a two-tailed Student t test. (B) On day 70 postimmunization, mice were sacrificed, and the brain and spinal columns were removed and fixed. The paraffin sections were stained with Luxol fast blue and H&E. Parenchymal inflammatory foci (.10 mononuclear cells) were quantified by a blinded neuropathologist, and results are presented as mean 6 SD per group. Statistical analysis was performed using a two-tailed Student t test. (C) Representative sections demonstrate more meningeal and parenchymal inflammation and larger areas of demyelination in the IL-11–treated versus the control mouse spinal cord. Luxol fast blue and H&E stain. Scale bar, 50 mm. The Journal of Immunology 5

IL-11 induces exacerbation of RREAE and increased whereas it did not change in other niches. Th17 cells in both com- numbers of IL-17A+CD4+ cells in the spinal cord partments expressed high levels of CCR6 (Fig. 5C). In contrast, the inflammatory infiltrates numbers of IFN-g+ and IL-4+CD4+ cells did not change (data not 6 The causal effect of IL-11 on the development of the Th17- shown). IL-11 also induced increased serum mean SD IL-17A mediated response was next examined in RREAE. Following levels in comparison with the control RREAE (Fig. 5D). We next studied the effect of IL-11 in the relapsing-remitting immunization of SJL/J mice with PLP139–151, the administration of recombinant mouse IL-11 for 10 consecutive days starting at the (RR) phase of the disease, which may be more relevant for human peak of the first relapse (day 12 postimmunization) induced an RRMS. A single dose of IL-11 was administered at day 56 post- + + acute exacerbation and increase in mean clinical scores that immunization and again increased the percentage of IL-17 CD4 remained elevated over the entire course of the disease (Fig. 4A). cells in the PBMCs, spleen, and spinal cord infiltrates within 16 h On day 70 postimmunization, the number of inflammatory foci (Fig. 5E). Studies of the adhesion molecules revealed that IL-11 was enumerated by a blinded neuropathologist and found to be administration during the RR phase of the disease increased + significantly higher (2.9-fold) in the spinal cord parenchyma of ICAM-1 expression (mean 6 SD) in brain-derived CD4 cells mice that received IL-11 in comparison with control RREAE (Fig. 5E). Gene expression studies at the same time point revealed (Fig. 4B) (13). increased IL-11 expression in the LN2s and increased IL-17A To test whether IL-11 induces a Th17 response in the acute phase expression (mean 6 SD) in the brain inflammatory infiltrates of disease, we injected a single dose of the recombinant mouse IL-11 (Fig. 5F). Serum cytokine measurements at the same time revealed (0.5 mg/mouse) or control vehicle (six mice per group) on day 12 increased IL-17A levels following IL-11 administration, in com- postimmunization. After 16 h, the mice were sacrificed, and cells parison with control RREAE (Fig. 5G). Interestingly, IL-11 ad- Downloaded from were harvested for further study. Flow cytometry studies revealed that ministration during the RR phase of the disease induced a IL-11 significantly increased the percentage of IL-17A+CD4+ cells in significant increase in ICAM-1 expression on IL-11+CD4+ cells PBMCs and in the spinal cord inflammatory infiltrates (Fig. 5A, 5B), from the brain and spinal cord as well as CCR6 expression on http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. IL-11 induces Th17 cell response in RREAE. (A) SJL/J mice were immunized with PLP139–151 peptide. On day 12 postimmunization, six mice per group were i.p. injected with recombinant mouse IL-11 (0.5 mg/mouse) or vehicle control. After 16 h, the mice were sacrificed and tissues were harvested. The cells were stimulated with PMA and ionomycin for intracellular staining, and the percentage of cells expressing each molecule was de- termined in gated CD4+ T cells. (B) Representative staining. (C) The percentage of CCR6-expressing cells in gated IL-17A+CD4+ cells. (D) The con- centration of IL-17A in serum samples was measured by ELISA. (E) On day 56 postimmunization, six mice per group received recombinant mouse IL-11 (0.5 mg/mouse) or control vehicle. After 16 h, the mice were sacrificed. The percentage of IL-17A+ cells was determined in gated CD4+ T cells from the PBMCs, spleen, and spinal cord inflammatory infiltrates. The percentage of ICAM-1+ cells was determined in gated CD4+ T cells from brain cell infiltrates. (F) The gene expression of IL-11 in LN and IL-17A in brain inflammatory infiltrates was detected by RT-PCR. (G) The concentration of IL-17A in the serum samples was measured by ELISA. (H) The percentages of ICAM-1+ cells were determined in gated IL-11+CD4+ T cells from the brain and spinal cord cell infiltrates. The percentage of CCR6+ cells was determined in IL-11+CD4+ T cells from the spinal cord infiltrates. The results are presented as mean 6 SD per group. Statistical analysis was performed using a two-tailed paired Student t test. Each experiment was performed once. 6 IL-11 INDUCES ENCEPHALITOGENIC Th17 CELLS spinal cord–derived IL-11+CD4+ cells (Fig. 5H), whereas the other In vitro studies of the CD19+ gated B cells demonstrated that IL-11 tested markers did not change (data not shown). induced the expansion of TNF-a+ and IL-1a+CD19+ cells and de- Ex vivo signaling studies revealed that a single IL-11 admin- creased the numbers of IL-10+ B regulatory cells (Fig. 6C), whereas istration during the RREAE induced a significantly increased the percentages of GM-CSF+,IL-23+,VLA-4+,IL-6R+, and IL-21R+ phosphorylation of NF-kΒ p65 and the expression of transcription B cells did not change in comparison with the control cultures. factor RORgt (Supplemental Fig. 1). These results are consistent Passive transfer of IL-11–induced encephalitogenic CD4+ cells with our study in CIS patients, in which IL-11 induced in a dose- induces RREAE dependent manner NF-kΒ p65 phosphorylation and RORc ex- pression, which were reversed by blocking aIL-11 mAb (Y. Tao, Passive transfer experiments were performed to detect whether + unpublished results). IL-11–polarized Ag-specific CD4 cells are able to induce RREAE in naive recipients. Separated CD4+ T cells from im- IL-11 induced encephalitogenic CD4+ cells in an in vitro Ag munized mice that were cultured with PLP139–151 in the absence or recall assay presence of IL-11 and control Th-17–polarizing cytokine IL-23 To test the hypothesis that IL-11 induces encephalitogenic CD4+ were injected i.p. in the naive mice. The mice that received IL-11 + cells, we cultured spleen- and LN-derived cells from PLP139–151- in vitro–polarized CD4 cells had more-severe disease in com- + immunized SJL/J mice with PLP peptide, in the absence or parison with the recipients of CD4 cells stimulated with PLP139–151 presence of IL-11 and the Th17-polarizing cytokine IL-23. IL-11 only (Fig. 7A). The difference in disease severity between the re- induced a significant increase in the percentage of IL-17A+CD4+ cipients of IL-11– and IL-23–polarized CD4+ cells was not statisti- cells, similar to IL-23, in comparison with the control Ag-stimulated cally significant. On day 70 post–passive transfer, the inflammatory Downloaded from cells. Ag recall in the presence of IL-11 also significantly increased foci were enumerated by a blinded neuropathologist and found to be the percentage of GM-CSF+, double-positive IL-17A+GM-CSF+,and significantly higher (1.5-fold) in the spinal cord meninges of mice IL-17A+IFN-g+CD4+ cells as well as IL-21+CD4+ cells (Fig. 6A), that received IL-11 in comparison with control RREAE (Fig. 7B). suggestive of the encephalitogenic phenotype of IL-11–polarized In a repeated experiment, the recipient mice were sacrificed at Ag-reactive CD4+ cells. To further characterize the IL-11–induced the peak of the disease (day 14), and flow cytometry studies were +

CD4 cell surface markers, we measured their expression of the performed on the PBMC, spleen, LN, and CNS-derived mono- http://www.jimmunol.org/ activation markers CD44, CD28, and PD-1; adhesion molecules nuclear cells. The results revealed that the recipients of IL-11– ICAM-1 and VLA-4; receptors CCR5 and CCR6; and polarized CD4+ cells had a higher percentage of IL-17A+CD4+ cytokine receptors IL-21R, IL-23, and IL-6R. Our data demonstrate that cells in LN- and CNS-derived mononuclear cells, a higher per- IL-11 induced the expansion of IL-6R+CD4+ cells (Fig. 6A), which may centage of CCR6+CD4+ cells in the CNS, and a higher percentage reflect their responsiveness to IL-6, a Th17-promoting cytokine, whereas of IL-17A+CCR6+CD4+ cells in the LN and spleen cells in the other tested marker did not change (data not shown). comparison with the control recipient mice (Fig. 7C), whereas In addition, we measured the capacity of IL-11 to induce the these markers did not change in the other niches. The numbers of expansion of CD8+ and CD19+ B cells. Studies of the CD8+ gated IL-11+, IFN-g+, and IL-21+ CD4+ cells did not change (data not + + cells revealed the expansion of IL-17A CD8 cells (Fig. 6B), shown). Studies of the adhesion molecules revealed an increased by guest on September 27, 2021 whereas their expression of IL-21, IL-23, VLA-4, and IL-6R was percentage of VLA-4+CD4+ cells in the PBMC and spleen cells not significantly changed (data not shown). of the recipients of IL-11–polarized Ag-specific CD4+ cells

+ FIGURE 6. IL-11 induced encephalitogenic CD4 cells. Spleen and LN cells were separated from PLP139–151-immunized SJL mice on day 11 postimmunization. Cells were cultured with PLP peptides (20 mg/ml) in the absence or presence of IL-11 (100 ng/ml) and the control Th-17–polarizing cytokine IL-23 (20 ng/ml) for 7 d. (A) Cells were restimulated with PMA/ionomycin for intracellular staining. The percentages of the cells expressing each molecule in gated CD4+ T cells (n =12),(B)CD8+ cells (n =6),and(C)CD19+ cells (n = 6) were determined by flow cytometry. Each symbol represents one mouse, and horizontal bars represent mean values. Statistical analysis was performed using repeated measures ANOVA. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 7. Passive transfer of IL-11–stimulated CD4+ cells induces severe RREAE. (A) CD4+ T cells were isolated from the cultures described in Fig. 6. A total of 20 3 106 CD4+ T cells per recipient were injected into naive SJL/J mice. Clinical scores of the six recipient mice per group were assigned daily for 70 d. Statistical analysis was performed using a repeated measures ANOVA. (B) On day 70 post–passive transfer, mice were sacrificed, and the brain and spinal cords were stained with Luxol fast blue and H&E. Meningeal inflammatory foci (.10 mononuclear cells) were quantified by a blinded neuropa- thologist, and statistical analysis was performed using a two-tailed Student t test. (C and D) Single-cell solutions were obtained from recipient mice sacrificed at day 14 post–passive transfer and incubated with PMA/ionomycin for intracellular staining. The percentages of cells expressing indicated by guest on September 27, 2021 markers in gated CD4+ cells, (E–G) viable cells, and (H) gated CD19+ B cells were determined by flow cytometry. (I) In mice sacrificed at day 70 post– passive transfer, the percentage of cells expressing the indicated markers in the viable cells and (J) gated CD4+ cells was determined by flow cytometry. The results represent mean 6 SD values per group. Statistical analysis was performed using paired t test. *p , 0.5.

(Fig. 7D), whereas the expression of ICAM-1 did not change (data whether CNS-derived cells from immunized mice that received not shown). IL-11 have enhanced encephalitogenic potential. Interestingly, the recipients of the IL-11–polarized CD4+ cells A total of 2 3 106 mononuclear cells were isolated from the also exhibited an expansion of neutrophils in the PBMC, spleen, brain and spinal cord tissue and transferred immediately to recipient and CNS-derived mononuclear cells (Fig. 7E), which was likely naive mice through i.p. injection. The recipients of cells from do- induced by IL-17A (15). In addition, transferring IL-11–polarized nors who received a single IL-11 dose 16 h prior to cell harvesting CD4+ cells caused the expansion of CD8a+ cells in LNs (Fig. 7F) had significantly higher mean 6 SD clinical scores in com- as well as the expansion of CD19+ cells in the CNS infiltrates parison with the mice that received CNS-derived cells from the (Fig. 7G) and increased percentage of IL-21R+CD19+ cells in LNs control RREAE donors (Fig. 8A). Passive transfer of CNS- (Fig. 7H). This may be related to the IL-11–induced IL-21 se- derived mononuclear cells from mice that received IL-11 in- cretion by CD4 cells, as demonstrated in the Ag recall assays duced a higher percentage of IL-17A+ and CCR6+CD4+ cells (Fig. 6A). in the recipients’ CNS in comparison with the control group In mice sacrificed on day 70 following passive transfer, flow (Fig. 8B). cytometry studies revealed a persisting increase of neutrophils in PBMCs (Fig. 7I) as well as an increased percentage of CCR6+CD4+ Discussion cells in the LNs of recipients of IL-11–polarized CD4+ cells, in We have previously reported that IL-11 and IL-17A CSF and serum comparison with the control recipient mice (Fig. 7J), whereas the levels are significantly increased in the CIS and RRMS patients in other markers did not change. comparison with the HCs. Serum levels of both cytokines are increased during the clinical relapses in comparison with the re- IL-11 enhances severity of the disease induced by passive missions of untreated RRMS patients (2). In the current study, we transfer of CNS-derived nonmanipulated mononuclear cells found that IL-11+CD4+ and IL-17A+CD4+ T cells accumulate in Because our data from mice with PLP139–151-induced RREAE the CSF of RRMS patients and in the active brain MS lesions, show that the percentage of Th17 cells in the CNS is significantly suggesting their role in the lesion formation. Following the pre- higher than in the peripheral lymphoid organs, we proposed to viously reported role of IL-11 in the in vitro differentiation and passively transfer CNS-derived cells to detect whether they can expansion of Th17 cells in early MS, our new findings suggest induce the disease even without in vitro polarization and to see that IL-11 may induce Th17 cell responses in vivo, which may 8 IL-11 INDUCES ENCEPHALITOGENIC Th17 CELLS

increased the numbers of spinal cord inflammatory foci, the numbers of peripheral and CNS-infiltrating IL-17+CD4+ cells, and IL-17A serum levels. In addition, IL-11 increased the numbers of ICAM-1+CD4+ T cells in the RREAE brain inflammatory infil- trates (2). ICAM-1 is an NF-kΒ–regulated adhesion molecule involved in the trans-endothelial migration to the CNS (25). ICAM-1–deficient mice showed attenuated EAE and dramatically reduced spinal cord T cell infiltrates. Furthermore, passive transfer of Ag-restimulated ICAM-12/2 T cells failed to induce EAE in wild-type mice (26). Finally, IL-11 mediated increased expression of CCR6 and ICAM-1 on IL-11+CD4+ T cells from CNS infil- trates, indicating that IL-11–induced CCR6 and ICAM-1 may mediate IL-11+CD4+ cell migration to the CNS. IL-6 and IL-11 induce inflammatory cell migration by regulating the expression of adhesion molecules and chemokine receptors. IL-6 induces VLA-4 expression on human CD4+CD45RO+ cells, and IL-11 directly mediates the in vitro migration of CD4+ cells from HCs (27). IL-11 is reported to induce VLA-2 and ICAM-1 ex-

pression in endometrial epithelial cells (28); however, its effect on Downloaded from brain endothelial cells (ECs) has not been examined. Finally, Th17 cell migration across the BBB-EC monolayer via IL-17R and IL-22R FIGURE 8. IL-11 enhances the disease severity induced by passive signaling is mediated via the disruption of tight junction proteins (29). transfer of nonmanipulated CNS mononuclear cells from mice with Both IL-17A (30) and IL-22 (31) induce IL-11 secretion, and our A RREAE. ( ) SJL/J mice that were immunized with PLP139–151 received previous results identified a positive correlation between IL-22 and 3 either IL-11 (0.5 mg) or control 1 PBS injection on day 11 post- IL-11 levels in the CSF of CIS patients (2). IL-17 also induces IL-6 http://www.jimmunol.org/ 6 immunization. The mice were sacrificed after 16 h. A total of 2 3 10 and neutrophil-attracting chemokine CXCL8 (IL-8) expression by mononuclear cells from the CNS infiltrate were isolated using a Percoll BBB ECs (32), which may enhance both neutrophil and CD4+ cell gradient and transferred to recipient mice via i.p. injection. Clinical scores migration through the BBB. Our results indicate that, similar to IL-6, of six recipient mice per group were assigned daily for 70 d. (B) On day 70 IL-11 induces CCR6 expression, which has been reported to mediate after passive transfer, CNS-infiltrating mononuclear cells of the recipi- + + ent mice were isolated by Percoll gradient. Cells were restimulated with IL-17A CD4 cell migration through the choroid plexus (3). PMA/ionomycin for intracellular staining. The percentages of CD4+ cells We have demonstrated that IL-11 induces encephalitogenic + expressing each molecule were determined by flow cytometry. The results CD4 cells, which induce RREAE upon passive transfer to naive represent mean 6 SD values. Statistical analysis was performed using repeated recipients. In Ag recall assays, IL-11 induced a significant ex- measures paired t test. Each experiment was performed once. *p , 0.05. pansion of IL-17A+, GM-CSF+, and IL-21+CD4+ cells. As stated by guest on September 27, 2021 in our previously published paper (2), IL-11 did not increase the expression of FOXP3 or the secretion of TGF-b or IL-10 in constitute an initial step in the development of the autoimmune FOXP3 memory CD4+ T cells. Because we did not detect IL-11– response in MS. mediated induction of T regulatory cells in CIS patients, we did Trafficking of the activated myelin-reactive T cells across the not pursue those studies in mice. Notably, a recent study (33) blood–brain barrier (BBB) into the CNS is a central event in the reported that IL-11 in Ag recall experiments from MBP- pathogenesis of MS (16–19). The migration of the inflammatory immunized mice induced Foxp3 gene expression, warranting cells through the choroid plexus and BBB is regulated by multiple future studies of the effect of IL-11 on T regulatory cells. Inter- adhesion molecules and chemokine receptors. In the RREAE estingly, IL-11 also induced the expansion of IL-17+CD8+ cells animal model of the disease, CCR6+ Th17 cells accumulate in the and increased the percentages of proinflammatory TNF-a+ and CNS, whereas passive transfer of CCR6-deficient CD4+ cells did IL-1a+B cells, whereas it decreased the numbers of regulatory not induce a disease, supporting a critical role of CCR6 in the IL-10+B cells. Passive transfer of IL-11–polarized Ag-specific early migration of inflammatory cells to the CNS (3). In vitro CD4+ cells into naive recipients induced severe RREAE, com- animal studies reveal that IL-11 induces IL-17A and CCR6 ex- parable to the prototypical encephalitogenic IL-23–polarized pression in CD4+ cells. In the same experiments, IL-11 induced CD4+ cells. Further characterization of the mechanisms involved STAT3 phosphorylation in a dose-dependent manner. Phosphory- in IL-11–polarized CD4+ cell–induced RREAE revealed increased lated STAT3 is a transcription factor that mediates IL-6–induced numbers of IL-17A+ and CCR6+CD4+ cells in both peripheral lym- Th17 cell differentiation and IL-23–mediated Th17 cell expansion phoid organs and the CNS and increased numbers of VLA-4+CD4+ (20). Furthermore, NF-kΒ c-Rel and RelA/p65 transcriptionally cells, which preferentially migrate to the CNS. Interestingly, transfer activate RORgt (21), which induces CCR6 expression in mouse of IL-11–polarized CD4+ cells also induces increased numbers of CD4+ cells (22). We therefore propose that the IL-11–mediated neutrophils in the periphery and the CNS, both acutely and in the induction of CCR6 expression may be regulated via RORc in- chronic phase of the disease, suggesting that IL-11 may contribute duced by STAT3 and p65 phosphorylation (21, 22). (similar to the IL-23/IL-17A/GM-CSF axis) to the regulation of IL-11 has been implicated in embryonal implantation (23) and neutrophil numbers in the peripheral circulation (34). We propose that metastatic cancer cell migration through IL-11R–induced NF-kΒ IL-11–induced expansion of neutrophils may constitute a key event in signaling (24). However, its role in the migration of inflammatory the IL-11–induced clinical worsening because the increased numbers cells across the BBB has not been examined. Our results indicate of neutrophils precede EAE clinical symptoms and neutrophil that administration of IL-11 at the time of clinical onset of blockade prevents the clinical onset of disease (15). The role of RREAE induced an acute exacerbation and increased clinical neutrophils in the opening of the BBB has been well established, scores, which persisted during the entire course of the disease. IL-11 as even their adhesion to the endothelial barrier may impair The Journal of Immunology 9 intraendothelial contacts via secretion of proteases and free 12. Zhang, X., Y. Tao, M. Chopra, M. Ahn, K. L. Marcus, N. Choudhary, H. Zhu, and S. Markovic-Plese. 2013. Differential reconstitution of T cell subsets fol- oxygen radicals. However, this study reports an IL-11–induced lowing immunodepleting treatment with Alemtuzumab (Anti-CD52 monoclonal neutrophil expansion. antibody) in patients with relapsing-remitting multiple sclerosis. J. Immunol. Further characterization of the inflammatory responses induced 191: 5867–5874. + 13. Sobel, R. A., V. K. Tuohy, Z. J. Lu, R. A. Laursen, and M. B. Lees. 1990. 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