IL-22 Binding Protein Promotes the Disease Process in Multiple Sclerosis Hannes Lindahl, André O

IL-22 Binding Protein Promotes the Disease Process in Multiple Sclerosis Hannes Lindahl, André O. Guerreiro-Cacais, Sahl Khalid Bedri, Mathias Linnerbauer, Magdalena Lindén, Nada This information is current as Abdelmagid, Karolina Tandre, Claire Hollins, Lorraine of September 24, 2021. Irving, Colin Glover, Clare Jones, Lars Alfredsson, Lars Rönnblom, Ingrid Kockum, Mohsen Khademi, Maja Jagodic and Tomas Olsson

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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 10, 2019, doi:10.4049/jimmunol.1900400 The Journal of Immunology

IL-22 Binding Protein Promotes the Disease Process in Multiple Sclerosis

Hannes Lindahl,* Andre´ O. Guerreiro-Cacais,* Sahl Khalid Bedri,* Mathias Linnerbauer,* Magdalena Linde´n,* Nada Abdelmagid,* Karolina Tandre,† Claire Hollins,‡ Lorraine Irving,x Colin Glover,‡ Clare Jones,‡ Lars Alfredsson,{ Lars Ro¨nnblom,† Ingrid Kockum,* Mohsen Khademi,* Maja Jagodic,* and Tomas Olsson*

Genome-wide association studies have mapped the speciﬁc sequence variants that predispose for multiple sclerosis (MS). The pathogenic mechanisms that underlie these associations could be leveraged to develop safer and more effective MS treatments but are still poorly understood. In this article, we study the genetic risk variant rs17066096 and the candidate gene that encodes IL-22

binding protein (IL-22BP), an antagonist molecule of the cytokine IL-22. We show that monocytes from carriers of the risk genotype Downloaded from of rs17066096 express more IL-22BP in vitro and cerebrospinal fluid levels of IL-22BP correlate with MS lesion load on magnetic resonance imaging. We confirm the pathogenicity of IL-22BP in both rat and mouse models of MS and go on to suggest a pathogenic mechanism involving lack of IL-22–mediated inhibition of T cell–derived IFN-g expression. Our results demonstrate a pathogenic role of IL-22BP in three species with a potential mechanism of action involving T cell polarization, suggesting a therapeutic potential of IL-22 in the context of MS. The Journal of Immunology, 2019, 203: 000–000. http://www.jimmunol.org/ ultiple sclerosis (MS), a chronic inflammatory disease revealed a large number of genetic variants that influence MS of the CNS, is still treated with relatively unspecific susceptibility (3–5). These are frequently found near immune- M modulators of the immune system (1, 2). Therefore, related genes, but the underlying mechanisms are, with few ex- lack of response and severe adverse events remain clinical chal- ceptions, unknown (6–10). Defining the causal genes and their lenges. Development of more appropriate treatments requires impact on MS pathogenesis could potentially lead to safer and pharmacological targeting of disease-specific molecular events more effective treatments. while sparing normal immune function. Drug targets that meet In the current study, we sought to clarify the role in MS of a risk these criteria can be identified by characterizing the genetic var- gene variant localized downstream of IL22RA2, namely, the single iants and, in extension, the physiological traits that predispose nucleotide polymorphism (SNP) rs17066096. This locus has re- by guest on September 24, 2021 for the disease. Recent genome-wide association studies have peatedly been associated with MS susceptibility (3–5) and encodes the IL-22 binding protein (IL-22BP), an antagonist molecule that tightly regulates the actions of IL-22 (11, 12). Signaling via the *Neuroimmunology Unit, Department of Clinical Neuroscience, Center for Molec- ular Medicine, Karolinska Institutet, 17177 Stockholm, Sweden; †Department of IL-22R, highly expressed on epithelia at barrier surfaces such as Medical Sciences, Rheumatology and Science for Life Laboratories, Uppsala Uni- skin, lungs, and gut, leads to tissue protection and regeneration as versity, 75105 Uppsala, Sweden; ‡Respiratory, Inflammation and Autoimmunity x well as induction of antimicrobial peptides (13, 14). Research, MedImmune, Cambridge CB21 6GH, United Kingdom; High Content Imaging, MedImmune, Cambridge CB21 6GH, United Kingdom; and {Institute of A relatively physiological way to characterize the effects of Environmental Medicine, Karolinska Institutet, 17177 Stockholm, Sweden IL-22 is to manipulate the levels of IL-22BP in animal disease ORCIDs: 0000-0002-4447-9426 (H.L.); 0000-0002-4561-2823 (A.O.G.-C.); 0000- models. We and others have shown that IL-22BP acts to limit 0002-7750-5723 (S.K.B.); 0000-0002-5528-5141 (M. Linnerbauer); 0000-0002- inflammation in models of psoriasis and inflammatory bowel 6951-526X (K.T.); 0000-0003-1688-6697 (L.A.); 0000-0003-0801-1444 (M.K.). disease, both affecting barrier surfaces (15, 16). In contrast, we Received for publication April 10, 2019. Accepted for publication June 18, 2019. have also shown that both rats with a natural genetic variant in this This work was supported by grants from the Swedish Research Council for Medicine locus that confers lower expression of IL-22BP and mice with full and Health (D0283001), King Gustav V’s 80-Year Foundation, the Knut and Alice Wallenberg foundation (2011.0073), the Swedish Brain Foundation, and AstraZenica/ gene deletion have less severe disease in an animal model of MS Science for Life. (17, 18). However, the role of IL-22 in MS is unclear. IL-22 is H.L., A.O.G.-C., M.J., and T.O. contributed to experimental design. K.T. and N.A. often described as a pleiotropic cytokine with a high degree of performed sample collection. H.L., A.O.G.-C., S.K.B., M. Linnerbauer, M. Lindeń, context dependency regarding its downstream effects (14). In- C.H., C.J., L.I., C.G., and M.K. performed experiments, data collection, and data analysis. H.L. wrote the manuscript. H.L., A.O.G.-C., S.K.B., M. Linnerbauer, M. Lindeń, flammation at barrier surfaces and in the CNS may thus be two N.A., L.A., L.R., I.K., M.K., M.J., and T.O. edited the manuscript. contexts in which the net effects of IL-22 are opposite. Address correspondence and reprint requests to Dr. Hannes Lindahl, CMM In this study, we show that an MS genetic risk sequence variant L8:04, Karolinska University Hospital, 17176 Stockholm, Sweden. E-mail address: strongly affected IL-22BP production and immune cell activation [email protected] state in vitro, as evidence for an expression quantitative trait locus The online version of this article contains supplemental material. (eQTL). We confirm the disease-promoting role of IL-22BP in rat Abbreviations used in this article: DA, Dark Agouti; DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; eQTL, expression quantitative trait locus; and mouse MS models and propose a mechanism of action whereby F, forward; IL-22BP, IL-22 binding protein; MOG, myelin oligodendrocyte glyco- IL-22 acts directly on T cells to limit their encephalitogenic po- protein; MS, multiple sclerosis; PVG, Piebald–Virol–Glaxo; qPCR, quantitative PCR; tential. Cytokines and their receptors, either soluble or membrane R, reverse; SNP, single nucleotide polymorphism. bound, are often considered as therapeutic targets in a variety of Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 inflammatory diseases. Our data on the IL-22 system provides

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900400 2 IL-22BP PROMOTES THE DISEASE PROCESS IN MS important background for any attempt to modulate this system as a hairpin RNA specific for Il22ra2 under the control of an H1 promoter with therapeutic strategy in humans. a tetO site as well as a TetR under the control of the CAGGS promoter was used (Fig. 3A). The DNA solution was microinjected into one cell stage fertilized embryos from DA/OlaHsd rats (Harlan) and transferred to oviducts Materials and Methods of pseudopregnant Sprague Dawley females. Integration of the transgene was Isolation of human PBMCs detected by PCR on genomic DNA isolated from ear biopsies with the primers 1165_48: 59-CCA TGG AAT TCG AAC GCT GAC GTC-39 and For experiments in Fig. 1F, buffy coats were collected from healthy blood 1165_46: 59-TAT GGG CTA TGA ACT AAT GAC CC-39. For knockdown donors for which rs17066096 genotype was known. PBMCs were isolated experiments, tap water was supplemented with 20–500 mg/l doxycycline using Ficoll-Pacque PLUS (GE Healthcare) gradient centrifugation. Buffy (D-9891; Sigma-Aldrich) and 1–10% (w/v) sucrose (Sigma-Aldrich) and coat cells were frozen in medium containing DMSO, later thawed to was administered ad libitum as drinking water. The solution was protected isolate CD14+ cells using MACS microbeads (Miltenyi Biotec), and then + from light and was freshly prepared every second day. live frozen again. CD14 cell samples from all healthy donors were thawed The mouse strains used for this research project, B6N.129S5- and plated in parallel to avoid batch effects. For experiments in Fig. 1A–D, Il22ra2tm1Lex/Mmcd (targeting Il22ra2), identification number 032407- blood was collected from healthy volunteers of unknown rs17066096 ge- UCD, and B6;129S5-Il22tm1.1Lex/Mmucd (targeting Il22), identification number notype and immediately processed for in vitro stimulation. 036745-UCD, were obtained from the Mutant Mouse Regional Resource In vitro stimulation Center, a National Center for Research Resources and National Institutes of Health–funded strain repository, and had been donated to the Mutant Mouse After thawing, CD14+ PBMCs were washed, counted, and resuspended at Resource and Research Center by Genentech (18). Il22ra2 is a constitutive a concentration of 1.5 3 106 cells per milliliter in RPMI 1640 medium knockout strain, and Il22 is a conditional knockout strain using the cre/loxP supplemented with 2 mM glutamine, penicillin, streptomycin, and 10% system. Because IL-22 is only expressed by hematopoietic cells, a full FBS. Two hundred microliters of cell suspension was added per well in 48- knockout is achieved by introducing cre recombinase under the Vav1 promoter. well plates. Aliquots of each sample were saved to assess purity of CD14+ To avoid bias, all mice used for experimental autoimmune encephalomyelitis Downloaded from isolation by flow cytometry, which was consistently .95%. Cells were (EAE) were bred to express Vav1-cre (The Jackson Laboratory). differentiated for 6 d with a change to fresh medium after 3 d. Cells were Female mice and rats between the age of 12 and 20 wk were used for all harvested by removing the medium and processing the cells for RNA experiments. Age-matched littermates were used as controls. Animals were isolation. The following reagents were used for in vitro differentiation of bred and maintained in a specific pathogen–free facility at the Karolinska CD14+ PBMCs: 400 IU/ml GM-CSF (PeproTech), 20 ng/ml IL-4 (R&D University Hospital (Stockholm, Sweden) in polystyrene cages with aspen Systems), 50 ng/ml M-CSF (R&D Systems), and 0.001–100 nM AM580 wood shavings, free access to water, and standard rodent chow.

(Sigma-Aldrich). To produce M1 and M2 polarized cells, M-CSF–differ- ELISA http://www.jimmunol.org/ entiated cells were additionally stimulated 24 h with fresh medium containing 100 ng/ml LPS + 20 ng/ml IFN-g and 20 ng/ml IL-4 (R&D Commercial ELISA kits were used to quantify human and rat IL-22BP Systems), respectively, with or without the addition of AM580. Because (Cloud-Clone Corp) and rat IL-22 (R&D Systems), according to the pro- AM580 is dissolved in DMSO, an equivalent amount of DMSO (the ve- tocol supplied by the manufacturer. Plasma and CSF were collected and hicle) was consistently added to wells that were not differentiated with stored at 280˚C until analysis. AM580. For activation of GM-CSF/IL-4–differentiated cells with TLR agonists in Fig. 1C, the following reagents were added 24 h before har- EAE vesting the cells: 1 mg/ml PAM3CSK4, 1 3 108 cells per milliliter of heat- killed Listeria monocytogenes,10mg/ml poly(I:C) high m.w., 10 mg/ml To induce EAE, rats were injected s.c. at the tail base with 4.5 mg of rat myelin poly(I:C) low m.w., 100 ng/ml LPS from Escherichia coli K12, 1 mg/ml oligodendrocyte glycoprotein (MOG) emulsiﬁed in Freund’s adjuvant

Flagellin from Salmonella typhimurium, 100 ng/ml FSL1, 10 mg/ml (Sigma-Aldrich). Mice were injected s.c. at the tail base with 50 mgofmouse by guest on September 24, 2021 Imiquimod, 10 mg/ml ssRNA40, and 5 mM ODN2006 (Invivogen). In MOG emulsified in Freund’s adjuvant supplemented with 200 mgnonviable Fig. 4B–D, CD4+ and CD8+ human PBMCs were isolated and plated at Mycobacterium tuberculosis H37 RA (Difco Laboratories). At immunization 80,000 cells per well in flat-bottom 96-well plates. Two microliters of and 2 d later, the mice were injected i.p. with 200 ng of pertussis toxin Dynabeads Human T-activator CD3/CD28 (Life Technologies) was added (Sigma-Aldrich). Animals were weighed and observed daily. Clinical signs to each well, according to the protocol supplied by the manufacturer. Cells were assessed as follows: 0, no paralysis; 1, tail paralysis; 2, hind limb were cultured 24 h prior to analysis by quantitative PCR (qPCR). Two paresis 3, hind limb paralysis; 4, hind and front limb paralysis; 5, dead. micrograms per milliliter of recombinant human IL-22 (R&D Systems) Rat lymph node cell isolation was added from the start, and cells were then cultured for 72 h followed by analysis by flow cytometry. For the experiment in Fig. 4E, blood samples Microbeads for OX-52 (T cells), CD45R (B cells), CD11b/c (myeloid cells), were collected from 84 individuals with MS or other neurologic diseases, and OX-62 (dendritic cells [DCs]) (Miltenyi Biotec) were used to isolate followed by PBMC isolation, live freezing, and storage in liquid nitrogen. basic cell types from single-cell suspensions of inguinal lymph node, Fifty thousand cells were plated in round-bottom 96-well plates with cell according to the protocol supplied by the manufacturer. culture medium alone or with the addition of 10 mg/ml LPS. Cells were harvested after 3.5 h and were processed for RNA extraction and qPCR Mouse CNS cell isolation for qPCR analysis. Fold change in expression was calculated by dividing relative Mice were perfused with PBS containing 500 ml/l Heparin (Leo Pharma). expression after LPS stimulation with relative expression in control wells. Brains were dissected and processed using the neural tissue dissociation kit DNA extraction and SNP genotyping (T) (Miltenyi Biotec), according to the protocol provided by the manufacturer. For magnetic separation of CNS cells, the following microbead Genotyping of healthy blood donors for the experiment in Fig. 1E and 1F kits for mice were used: ACSA-1, ACSA-2, O4, CD11b, and CD171 was performed on the ImmunoChip (Illumina) by the SNP&SEQ Tech- (Miltenyi Biotec). A dedicated CNS cell sample fraction was used to nology Platform, Science for Life Laboratory and the National Genomics isolate CD11b+ microglia by positive selection. To sort astrocytes, another Infrastructure at Uppsala University. For the experiment in Fig. 4E, total CNS cell sample fraction was incubated with a pool of ACSA-1 and DNA was extracted from blood samples using QIAamp Blood Maxi kit ACSA-2 microbeads followed by positive selection. The flow-through (Qiagen), according to the protocol provided. rs17066096 genotype was from this step was then passed through an LD column (Miltenyi Biotec) assessed using the TaqMan OpenArray Genotyping System (Applied to achieve an astrocyte-depleted fraction, which was split into two frac- Biosystems). tions for positive selection of O4+ oligodendroglia and CD171+ neurons. Purity was assessed by qPCR of Gfap, Mog, Itgam (CD11b), and Rbfox3 Rat and mouse strains (NeuN) (Supplemental Fig. 3B). The Dark Agouti (DA) rat strain was originally obtained from the Oligodendrocyte progenitor cell culture Zentralinstitut fu¨r Versuchstierzucht (Hannover, Germany), and MHC- identical Piebald–Virol–Glaxo (PVG.1av1, referred to as PVG in this ar- Oligodendrocyte progenitor cells were purified by magnetic cell sorting. Brains ticle) was obtained from Harlan UK Limited (Blackthorn, U.K.). Colonies were obtained from postnatal day 2 Wistar rats (Charles River), and the have thereafter been established at Karolinska University Hospital (DA/ forebrains were removed by crude dissection. Tissue was diced and digested in Kini and PVG.1av1/Kini). An inducible knockdown rat strain on the DA papain (Worthington) at 37˚C for 60 min and then gently dissociated. Dis- background was generated by TaconicArtemis, project name DA/OlaHsd- sociated postnatal day 2 brains were incubated with 20 ml A2B5-coated Tg(H1/tetO-RNAi:IL22ra2)Arte. A DNA construct containing a short microbeads (Miltenyi Biotec) per 107 cells for 15 min at 4˚C. After The Journal of Immunology 3 rinsing with DMEM containing 13 ITS supplement (Invitrogen), up to Total RNA was extracted using the RNeasy mini kit (Qiagen) with the 108 dissociated cells were resuspended in 3 ml of DMEM/ITS and ap- QIAcube (Qiagen), including on-column digestion of DNA using the pliedtoanLSmagneticseparationcolumnplacedinamagneticfield. RNase-free DNase set (Qiagen). The iScript cDNA synthesis kit (Bio-Rad) After washing the column with 3 ml of DMEM/ITS, the positively se- was used to prepare cDNA. Real-time RT-PCR was performed on a CFX384 lected A2B5+ cells were eluted from the column by removing the column Real-Time system machine (Bio-Rad) using IQ SYBR green Supermix from the magnetic field, passing 5 ml of complete media (see below) (Bio-Rad). Cycling conditions were as follows: 95˚C for 3 min, (95˚C for through, and collecting the eluted cells in a collection tube. A2B5+ cells 15 s, 60˚C for 30 s, 72˚C for 30 s) (40 cycles), 95˚C for 10 s, followed by melt were transferred to poly-D-lysine–coated 96-well culture plates and curve analysis. Data were analyzed manually using the D cycle threshold seeded at 10,000 cells per well. All cells were cultured at 37˚C, 5% CO2 method with HPRT or Hprt as reference. Primer sequences used are as in DMEM containing penicillin–streptomycin (100 U each), 13 Gluta- follows: human HPRT: forward (F): 59-ACCAGTCAACAGGGGACATAA-39, MAX supplement, 13 N2 supplement (all from Invitrogen), and modi- reverse (R): 59-CTTCGTGGGGTCCTTTTCACC-39;humanIL22RA2:F:59- fied NS21 (19) including 2.5 mg/ml BSA, 2.5 mg/ml catalase, 1 mg/ml ACACTTGCAACCATGATGCC-39,R:59-TGAGGCTTCAGAGACTCATGC- glutathione, 2.5 mg/ml superoxide dismutase, 2 mg/ml L-carnitine, 1 mg/ml 39; human IL22RA1:F:59-AGATATGTCACCAAGCCGCC-39,R:59- ethanolamine, 15 mg/ml D-galactose, 1 mg/ml linoleic acid, 1 mg/ml lino- CCGCTGAGGTCAAAGACAGG-39;humanIL22:F:59-CCAGCATG- lenic acid, 0.047 mg/ml Lipoic acid, 1 mg/ml tocopherol, 1 mg/ml tocopherol AAGGTGCGGTTGGT-39,R:59-TGCGCCCATCAGCTCCCACT-39;rat acetate, and 10 nM hydrocortisone. The following day, the cells were treated Hprt:F:59-CTCATGGACTGATTATGGACA-39,R:59-GCAGGTCAG- with differentiation factors 10 ng/ml T3 (Sigma-Aldrich) and 10 ng/ml CNTF CAAAGAACTTAT-39;ratIL22ra2:F:59-CAACCACGATGCCTAAGC-39, (R&D Systems) with or without 500 ng/ml recombinant rat IL-22 (R&D R: 59-CAACCACGATGCCTAAGC-39;ratIl22ra1:F:59-TACACGTGCC- Systems). Cells were cultured for a further 6 d with feeding every 2–3 d. GAGTGAAGAC-39,R:59-GCTGGACATTCAGGGAGTTAGG-39;ratIl22: F: 59-CAGGAGGTGGTGCCTTTCC-39,R:59-TCTTCTGGATGTTCTGG- Immunostaining TCATCA-39; mouse Hprt:F:59-ACAGCCCCAAAATGGTTAAGG-39, R: 59-TCTGGGGACGCAGCAACTGAC-39; mouse Il22ra2:F:59-GA-

Cells were ﬁxed with 4% formaldehyde (Pierce) for 20 min at room AGGTCCGATTTCAGTCCA-39,R:59-TCACCCTCCCGTAATACAGC- Downloaded from temperature. After washing, cells were permeabilized with 0.1% Triton 39; mouse Il22ra1:F:59-GCTCGCTGCAGCACACTACCA-39,R:59- X/PBS for 5 min. Cells were stained with HCS cell mask green (Invitrogen) TCCAGGGTTAGCTGGTGGCCA-39; and mouse Il22:F:59-GCTCAG- before blocking with 3% BSA (Sigma-Aldrich) in PBS and staining with CTCCTGTCACATCA-39,R:59-CAGTTCCCCAATCGCCTTGA-39. Hoechst 1:10,000 (Invitrogen), anti-MBP 1:1000 (Abcam), and goat anti- mouse secondary Ab Alexa Fluor 647 1:1000 (Invitrogen). Cells were Flow cytometry imaged on the OPERA QEHS using 103 air objective and analyzed using Columbus software. Rat inguinal lymph nodes were collected in cell culture medium and passed

through a 40-mm strainer to make single-cell suspensions. Cells were in- http://www.jimmunol.org/ Isolation and culturing of mouse astrocytes cubated with yellow dead cell staining (Invitrogen) and stained with following Abs: FITC CD3 (clone IF4; BD Biosciences), PE IFN-g (clone Brains of PBS-perfused wild-type mice were aseptically dissected and collected DB-1; BD Biosciences), PerCP/Cy5.5 IL-17A (clone eBio17B7; Thermo in 50-ml tubes containing 10 ml of ice-cold HBSS, then transferred into 5 ml of Fisher Scientific), PE-Cy7 Foxp3 (clone FJK-16s; Thermo Fisher Scien- Leibovitz’s L15 medium (Thermo Fisher Scientific) containing 500 U of Pa- tific), and APC CD4 (clone OX-35; BD). Human PBMCs were isolated pain (Worthington) per brain and mechanically minced by pipetting up and and used for in vitro experiments, as described, and were stained with the down through a 25-ml pipette. The homogenate was incubated in a water bath following Abs: PE/Dazzle CD3 (clone OKT3; BioLegend), Alexa Fluor at 37˚C for 15 min. After 5 min of incubation, remaining tissue pieces were 700 CD4 (clone A161A1; BioLegend), Alexa Fluor 700 CD8a (clone further reduced by trituration with 1-ml tips. After 10 min of incubation, tissue HIT8a; BioLegend), PerCP/Cy5.5 IL-17A (clone BL168; BioLegend), PE was triturated by pipetting with a 200-ml tip prior to addition of 400 U of IFN-g (clone B27; BioLegend), APC Foxp3 (clone PCH101; Thermo DNase I (Roche). The enzymatic reaction was stopped by adding 20 ml of Fisher Scientific), and V450 Ki-67 (clone B56; BD Biosciences). Foxp3 by guest on September 24, 2021 HBSS + 10% FBS (v/v). The homogenate was transferred and filtered through staining kit (Thermo Fisher Scientific) was used for intracellular staining. a40-mm cell strainer into a new 50-ml tube. After centrifugation at 200 3 g, Abs used for spinal cord–infiltrating cells in mice were as follows: PE-Cy7 the pellet was resuspended in 20 ml of 20% Percoll (Sigma-Aldrich) in HBSS CD45.2 (clone 30-F11; BioLegend), PerCP/Cy5.5 CD11b (clone M1/70; (v/v) and centrifuged for 20 min at 200 3 g at room temperature with low BioLegend), V500 CD3 (clone 500A2; BD Biosciences), FITC CD11c acceleration and no break. Following density centrifugation, the myelin layer (clone HL3; BD Biosciences), V450 Ly6G (clone 1A8; BD Biosciences), and remaining supernatant were removed using a 25-ml pipette. The pellet was and near-IR dead cell staining dye (Invitrogen). All samples were acquired washed in HBSS at 200 g. A pellet of one to three brains containing mixed glia on a Gallios flow cytometer (Beckman Coulter). Data were analyzed using cells was resuspended in 20 ml of prewarmed DMEM/F12 (Thermo Fisher the software Kaluza Flow Cytometry Analysis v1.1 (Beckman Coulter). Scientific) including 10% FBS (v/v), 100 U per ml/100 mg per ml penicillin/ streptomycin (Sigma-Aldrich), and 20 ng/ml mouse M-CSF (R&D Systems) Statistics andseededintoanuncoatedT75cellculture flask. The medium was changed The statistical tests used are described in the figure legends along with twice a week until the cells reached 90% confluency (12–16 d). descriptions of measures of central tendency and dispersion as well as To obtain astrocytes, mixed glial cells were detached by 10 ml of pre- correction for multiple testing. The a value was set to 0.05 throughout the warmed Trypsin-EDTA 0.05% (Thermo Fisher Scientific). After 10–15 study. The software used was Prism 5 (GraphPad Software). min of incubation in Trypsin-EDTA at 37˚C, cells were collected through a 40-mm cell strainer into a 15-ml Falcon tube, and the reaction was Study approval stopped by adding 5 ml of pure FBS. The single-cell suspension containing both microglia and astrocytes was centrifuged at 300 3 g for 10 The collection and use of human samples were approved by the Regional min. The mixed glia culture was depleted of CD11b+ cells using magnetic Committee of Ethics (Regionala Etikpro¨vningsna¨mnden, Stockholm and beads (Miltenyi Biotec). Cells in the negative fraction were resuspended at Uppsala) and were performed in accordance with the declaration of a density of 105 cells per ml in DMEM/F12 including 10% FBS (v/v) and Helsinki. Written informed consent was obtained from each human penicillin/streptomycin and seeded at 1 ml per well in an uncoated 24-well subject that contributed samples for this study. All procedures performed plate (Thermo Fisher Scientific). After 24 h, cells were stimulated in on animals in this study were approved by the local ethics committee prewarmed complete medium containing either 100 ng/ml LPS (Sigma- (Stockholms norra djurfo¨rso¨ksetiska na¨mnd). Aldrich), 20 ng/ml IFN-g (R&D Systems), 30 ng/ml TNF (R&D Systems), 3 ng/ml IL-1a (R&D Systems), or 1 mM H2O2 or medium without FBS (for starvation) alone or in the presence of 10 ng/ml IL-22 (R&D Systems) for 48 h, Results after which cells were detached with prewarmed Trypsin-EDTA 0.05%; IL22RA2 is not expressed by circulating immune cells but stained with APC Annexin V and propidium iodide (BioLegend), according to is highly induced in monocytes differentiated in vitro with the manufacturer’s protocol; and quantified using flow cytometry. GM-CSF, IL-4, and retinoic acid receptor agonist AM580 RNA isolation and qPCR IL22RA2 is the nearest gene, and thus candidate gene, for the MS risk locus on chromosome 6 identified by an observed case-control Cells were disrupted in RLT buffer (Qiagen) with 1% 2-ME and homogenized by repeatedly passing the lysate through a needle with an RNase-free segregation of allele frequencies of SNP rs17066096 (3–5) syringe. Tissue samples were disrupted and homogenized with RNase-free (Supplemental Fig. 1). We hypothesized that rs17066096 geno- steel spheres in RLT buffer with 1% 2-ME using a TissueLyzer LT (Qiagen). type influences MS risk by altering transcription of IL22RA2 and 4 IL-22BP PROMOTES THE DISEASE PROCESS IN MS set out to investigate this in human peripheral blood cells. No rs17066096 genotype was assessed at the end of the experiment. eQTLs for this variant are indicated in the Genotype-Tissue Ex- In addition to baseline expression in the monocyte-derived DCs, pression portal database (gtexportal.org). Although transcription we also included three more conditions to study the effect of of IL22 and IL22RA1 was detected in human peripheral blood rs17066096 genotype on IL22RA2 expression in DCs differentiated cells, IL22RA2 transcription was not (Fig. 1A). However, in the in the presence of AM580 and those that also had been matured Genotype-Tissue Expression data, occasional whole blood sam- with LPS stimulation. IL22RA2 expression was significantly higher ples have measurable quantities of IL22RA2, although median in cells from the individuals carrying the MS-associated genotype expression is 0 (gtexportal.org). To study the link between rs17066096GG when cultured with 1 nM of AM580 without or with rs17066096 genotype and expression levels of IL22RA2,we 24 h of LPS stimulation (Fig. 1F). This is consistent with the MS therefore first had to set up a suitable expression system in vitro. risk SNP rs17066096 tagging an eQTL that under specific cir- This has the added advantage of enabling us to skew the cells with cumstances affects expression of nearby gene IL22RA2. different stimuli, knowing that the effect of an SNP on tran- IL-22BP in CSF correlates with magnetic resonance imaging scription may appear only under certain conditions (20). IL22RA2 lesion load in MS patients is known to be secreted by APCs in tissues (18, 21). Therefore, peripheral blood monocytes were isolated from healthy individu- Although IL22RA2 transcription is not detectable in circulating als and differentiated into APCs with similarities to macrophages cells, we detected the protein product in both plasma and CSF by and DCs. Culturing the monocytes for 6 d with IL-4 or the ELISA. In vitro, we only saw a significant effect of rs17066096 combination of GM-CSF and IL-4 induced prominent expression genotype on IL22RA2 expression when the cells had been con- of IL22RA2 (Fig. 1B). Further, considerable amplification of ditioned with retinoic acid receptor agonist AM580. Accordingly, Downloaded from IL22RA2 expression was achieved when the retinoic acid receptor no significant associations to rs17066096 genotype were observed agonist AM580 was included from the start of the differentiation in relation to levels of IL-22BP (Fig. 1G). Next, we sought to link protocol, with a maximum effect in combination with GM-CSF IL-22BP levels in plasma and CSF with measures of MS severity, and IL-4. Similar results have been reported by others (21). The so we used the Expanded Disability Status Scale and lesion effect of AM580 on IL22RA2 expression in GM-CSF– and IL-4– number on magnetic resonance imaging. In our database, lesion

differentiated monocytes was dose dependent with a maximum number is categorized on a 5-level scale (0, 1–2, 3–5, 6–8, and 9 http://www.jimmunol.org/ around 10 nM (Supplemental Fig. 2A). It has been shown previ- or more lesions), with the highest level being more common than ously that stimulation in vitro with LPS decreases expression of the other levels combined. For this reason, a cut-off was made at IL22RA2 in monocyte-derived DCs (16). We sought to confirm nine lesions to define low and high lesion load. Patients with high this and contrast it with the effects of signaling via the other TLR. lesion load had significantly higher levels of IL-22BP in the CSF Again, using monocytes differentiated with GM-CSF and IL-4 as (Fig. 1H). No significant correlation between IL-22BP in CSF and baseline, dramatic reduction of IL22RA2 expression was achieved age or Expanded Disability Status Scale was observed (Supplemental by LPS when added during the final 24 h of differentiation (Fig. Fig. 1B), suggesting that IL-22BP covaries with inflammation rather 1C), even in the context of AM580-differentiated cells (Fig. 1D). than neurodegeneration. Varying degrees of reduction were also seen by the other tested by guest on September 24, 2021 TLR ligands (Fig. 1C). In summary, IL22RA2 mRNA is not de- Expression levels of IL-22BP and IL-22 follow the disease tected in circulating cells but can be highly induced in vitro course in an experimental MS model by GM-CSF, IL-4, and AM580, resulting in a phenotype that To generate hypotheses regarding the role of the IL-22 system in has similarities with tissue APCs. Addition of established APC autoimmune neuroinflammation, we studied how the expression of activation stimuli, TLR ligation, results in reduced IL22RA2 its components varied over the course of MS model EAE. The EAE expression. model in C57BL/6 mice is widely used to study pathophysiological

G mechanisms relevant for MS. However, MOG-induced EAE in DA MS susceptibility variant rs17066096 is associated with rats has some distinct advantages over the standard mouse model. higher IL22RA2 expression in differentiated monocytes Most notably, it requires a milder adjuvant for disease induction, Next, we set out to investigate the impact of MS-associated SNP presents with a relapsing remitting disease course similar to MS, rs17066096 on IL22RA2 transcription. To avoid confounding and the larger animal size makes it easier to sample CSF for factors such as severity of disease and inflammation, disease stage analysis (22, 23). Moreover, we have previously demonstrated a and type, as well as the wide range of different MS treatments, similar association of the homologous Il22ra2 gene with risk and we chose to proceed with samples from healthy individuals. We severity of rat EAE (17). Samples were collected at fixed intervals collected blood samples from healthy donors, all preselected before onset and at clinically predefined stages after onset of based on the rs17066096 genotype. Preselection was necessitated disease. In these experiments, onset of EAE was relatively late, because the population frequency of the risk GG genotype is only after day 15. Tissue samples from the inguinal lymph nodes, ∼5%. Individuals homozygous for each allele were included. which drain the site of MOG immunization, were collected from Following mononuclear cell extraction, the monocytes were iso- DA rats as well as PVG rats that are resistant under the same lated and then frozen in medium containing DMSO for later use. induction protocol. Expression of the Il22 gene increased mod- When the collection phase was completed, cells from all indi- erately in both strains but more so in the DA strain, with a max- viduals were thawed and differentiated in parallel. The protocol imum around day 7, which corresponds to the peak of immune cell used for these experiments involved differentiation of monocytes activation (Fig. 2A). The expression pattern of the Il22ra2 gene in the presence of GM-CSF and IL-4 for 6 d to induce a phenotype was essentially inverted compared with that of Il22. Low levels of similar to DCs (Fig. 1E). The purpose of using so-called Il22ra1 were detected, which increased slightly over time. Next, monocyte-derived DCs in this study is not to draw conclusions draining lymph nodes were collected from DA rats that were about the behavior of bona fide DCs in vivo but rather to inves- untreated (day 0) or 7 d after immunization, followed by enzy- tigate the potential of this genetic variant to influence gene matic digestion and isolation of basic cell types. The Il22 gene expression in the monocyte lineage after exposure to plausi- mRNA was detected in all cell types (Fig. 2B). Interestingly, cell ble physiological stimuli. IL22RA2 expression in relation to samples that had been depleted of most T cells, B cells, CD11b/c+ The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. rs17066096GG genotype is associated with higher IL22RA2 expression in monocytes differentiated with GM-CSF, IL-4, and retinoic receptor agonist AM580. (A) Mononuclear cells and granulocyte fraction from human healthy donor (n = 2) peripheral blood were sorted into basic cell subsets, and expression of IL22, IL22RA1,andIL22RA2 was assessed by qPCR (mean 6 SEM). IL22RA2 was not detected at all in these samples. (B) Monocytes from healthy donors (n =2) were differentiated into a panel of in vitro models of APC, with and without inclusion of retinoic acid receptor agonist AM580, and IL22RA2 expression was assessed by qPCR (mean 6 SEM). (C) TLR agonists were added to GM-CSF– and IL-4–differentiated healthy donor (n = 2) monocytes 24 h before harvesting the cells, and expression of IL22RA2 was assessed by qPCR (mean 6 SEM normalized to baseline, no TLR). TLR1/2, Pam3CSK4; TLR2, heat-killed L. monocytogenes; TLR3A, poly(I:C) high m.w.; TLR3B, poly(I:C) low m.w.; TLR4, LPS (20 ng/ml); TLR5, Flagellin; TLR6/2, FSL1; TLR7, Imiquimod; TLR8, ssRNA40; and TLR9, ODN2006. (D)Sameasin(C) but with 10 nM of AM580 included during differentiation and testing different concentrations of LPS. Data in (A)–(D) are compiled from two independent successive experiments. (E and F) Monocytes were collected from healthy donors with known rs17066096 genotype (AA, n =8;GG,n = 10) and were differentiated with GM-CSF and IL-4 alone or with the addition of 0.1 nM AM580 or 1 nM AM580 without and with the addition of 20 ng/ml of LPS added 24 h before harvesting the cells. IL22RA2 expression was assessed (mean 6 SEM) and comparisons between groups were performed using unpaired two-tailed Student t test, and p values are Bonferroni corrected for four tests. (G) IL-22BP levels were determined by ELISA in plasma and CSF from MS patients, and the results were stratified according to rs17066096 genotype (plasma: AA, n =12;AG,n =11;GG,n = 3; CSF: AA, n =22;AG,n = 20; GG, n =14; median 6 interquartile range (IQR). (H) MS patient plasma and CSF levels of IL-22BP were assessed for correlation with magnetic resonance imaging lesion load, which is categorized in the database on a 5-level scale (0, 1–2, 3–5, 6–8, and 9 or more lesions). Low (,9 lesions; plasma: n =21;CSF,n = 20) is compared with high (plasma: n =73;CSF,n = 61), and results are median 6 IQR. Significance testing for IL-22BP levels in plasma and CSF has been performed using Mann– Whitney U test because of deviation from normality. Both have p , 0.0001 using the D’Agostino and Pearson normality test. cells, and OX-62+ DCs (called marker negative in this article) had IL-22 is therefore likely low. The increased Il22 expression seen in the highest expression of Il22. However, the marker-negative cells Fig. 2A is likely to primarily reflect the recruitment of IL-22– are relatively few, and their contribution to the total amount of producing cells rather than increased production from resident 6 IL-22BP PROMOTES THE DISEASE PROCESS IN MS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. Expression levels of IL-22BP and IL-22 follow clinical course in MS model EAE. (A) EAE-susceptible DA rats and the relatively resistant PVG rats were immunized with MOG(1–125) in IFA. Lymph nodes were collected at fixed time points before onset of paralysis and at predefined stages of disease after onset. Ons, first day of tail paralysis; peak, first day of complete paraparesis; and rem, first-day motor improvement was observed in at least one of the hind limbs after having had an EAE score of at least 3. (n $ 3 per strain and time point.) Expression of Il22, Il22ra2,andIl22ra1 was determined by qPCR (mean 6 SEM). (B) Lymph nodes from naive (day 0) and EAE-immunized (day 7) wild-type DA rats (n $ 2 per time point) were digested to recover both lymphocytes and APCs. OX52+ Tcells,CD45R+ B cells, CD11b/c+ myeloid cells, OX-62+ DCs, and cells negative for all four markers were sorted using magnetic beads, and expression of Il22, Il22ra2,andIl22ra1 was determined using qPCR (mean 6 SEM). (C) Wild-type DA rats were immunized with MOG to induce EAE, and spinal cord, plasma, and CSF samples were collected at similarly predefined time points (n = $ 3). Expression of Il22, Il22ra2,andIl22ra1 was determined in spinal cord tissue by qPCR (white bars, mean 6 SEM). Significance testing was performed using Mann–Whitney U test for comparing the various timepoints with baseline expression or Kruskal–Wallis test for comparing expression in sorted cells. Only significant differences are indicated (by an asterisk [*]), in which *p , 0.05. CSF levels of IL-22 and IL-22BP were quantified by ELISA, and they are presented on an inverted scale (black bars) below the corresponding transcript in spinal cord samples. CSF samples (n $ 3) were pooled groupwise prior to analysis because of the small volumes obtained. cells. Consistent with the human data in Fig. 1, we observed rats, which was positively correlated with a trend toward increased the highest expression of the Il22ra2 transcription in APCs and CSF IL-22BP levels at the peak of disease that decreased as motor a reduction upon activation. Interestingly, the IL-22R subunit function was regained. IL-22R is not possible to measure by Il22ra1, which is often reported as absent on immune cells, ELISA, but transcription of the receptor subunit Il22ra1 was de- became detectable in one T cell–enriched sample 7 d after tected in the spinal cord with a trend toward increased expression immunization. throughout the disease course. To further characterize the cellular The expression and role of the IL-22 system components in the sources of Il22, Il22ra2, and Il22ra1 in the CNS, their expression CNS is even less well defined. In samples collected in a similar was determined in mouse brain samples. The choice of species manner, transcripts and protein levels of IL-22 were transiently was motivated by better availability of cell-specific markers for increased in the spinal cord and CSF during the onset of disease sorting in the mouse. Samples from naive brains and brains taken (Fig. 2C). We observed a slight increase in Il22ra2 transcription in 29 d after EAE induction were dissociated and enriched for the spinal cord during all stages of disease compared with naive astrocytes, oligodendroglia, microglia, or neurons. Interestingly, The Journal of Immunology 7

Il22ra2 was detected in CD11b+ cells enriched for microglia mediated by short hairpin RNA (Fig. 3A, 3B, Supplemental (Supplemental Fig. 3A). Moreover, Il22ra1 mRNA was detected Fig. 4A, 4B). With this approach, the rats have normal Il22ra2 in O4+ oligodendroglia and, to a lesser extent, in astrocytes. IL-22 expression until the start of doxycycline administration, thus has a well-documented tissue-protective role in several organs avoiding potential compensatory changes during development. outside the CNS (24). A report has also shown that human primary Starting doxycycline treatment at 9 d after EAE induction, which astrocytes exhibit less stress-induced apoptosis if IL-22 is added in knocks down Il22ra2 expression after the priming events have the medium (25). In light of this and our previous data, we hy- taken place, was partially protective in this model but required pothesized that IL-22 may have a proliferative or protective effect 500 mg/l of doxycycline (Fig. 3C). However, knocking down on these glial cells. To test this, rat oligodendrocyte progenitor Il22ra2 expression before immunization had a dose-dependent cells were differentiated in vitro with the addition of recombinant inhibitory effect on EAE, reducing severity with 4 and 20 mg/l rat IL-22, but no effect on differentiation or cell numbers was of doxycycline and making the transgenic rats completely resistant observed (Supplemental Fig. 3C). In the same assay, astrocyte to disease with 100 mg/l (Fig. 3D), a dose that had no effect when density could be assessed, but no effect was seen by IL-22 stim- administration started 9 d after immunization (data not shown). ulation. We also treated mouse astrocytes with a panel of stressors These ﬁndings further support a role of IL-22BP during priming in with or without addition of IL-22 followed by Annexin V staining, the periphery rather than during the effector phase or the response but no effect on apoptosis was observed (Supplemental Fig. 3D). in the CNS. In summary, the expression patterns of IL-22 and IL-22BP are IL-22BP promotes the Th1 response dynamically regulated over the course of EAE in rats, both in the lymph nodes where the immune response is initiated as well as in The data so far indicate potential roles for IL-22BP in both the Downloaded from the CNS. IL-22R can be detected in both compartments but, periphery and in the target organ. However, expression of IL-22BP contrary to Perriard et al (25), we could not demonstrate a trophic is much higher in secondary lymphoid tissue (Supplemental Fig. effect of IL-22 on astrocytes or on oligodendrocytes. 4B), and the effect of knocking down gene expression before immune cell activation has taken place is dramatic (Fig. 3D). Inducible IL-22BP knockdown in the rat species reduces Moreover, no effect of IL-22 on cultured glial cells could be susceptibility and severity of EAE

demonstrated (Supplemental Fig. 3C, 3D). Keeping in mind that http://www.jimmunol.org/ The human data suggest that genetically determined changes in an effect of IL-22 in CNS during EAE cannot be formally ex- IL22RA2 levels influence susceptibility and possibly also severity cluded, we now focus on the periphery in which the IL-22 system of MS (Fig. 1). We therefore hypothesized that moderate changes clearly is involved. Hypothesizing an effect of IL-22BP on the in Il22ra2 expression will influence susceptibility to or severity of initiation of the immune response in EAE, we focused on T cells rat EAE by acting in the draining lymph node or in the CNS. For in the draining lymph nodes 7 d after immunization. This is further this purpose, we developed an inducible Il22ra2 knockdown rat justified by our observations of IL22RA1 expression in human strain in which administration of doxycycline in the drinking T cells ex vivo (Fig. 1A) and upregulation of Il22ra1 expression in water results in a dose-dependent decrease in Il22ra2 expression, draining lymph nodes 7 d after MOG immunization in rats (Fig. by guest on September 24, 2021

FIGURE 3. Inducible knockdown of Il22ra2 in vivo has a dose-dependent effect on EAE disease course. (A) The transgenic vector used to generate a DA rat strain that enables inducible knockdown of Il22ra2 using a tetracycline-controlled gene expression system. (B) Knockdown efﬁ- ciencies in a panel of tissues (n = 3 per group, mean 6 SEM) after treatment with 100 mg/l of doxycycline for 10 d, which leads to reduced Il22ra2 expression in rats carrying the transgene (TG) compared with wild-type (WT) rats. (C) 500 mg/l of doxycycline was added to the drinking water of all rats starting from day 9 after EAE immunization (n = 10 in each group, mean 6 SEM). (D) Il22ra2 knockdown was induced 4 d prior to EAE immunization us- ing4mg/l(WT,n =9;TG,n = 11), 20 mg/l (WT, n =8;TG,n = 8), and 100 mg/l (WT, n = 11; TG, n = 10) of doxycycline in the drinking water. Incidence for each group is reported as a percentage in the respective plot. Signiﬁcance testing was performed using Mann–Whitney U test, in which *p , 0.05, **p , 0.01. dox, doxycycline. 8 IL-22BP PROMOTES THE DISEASE PROCESS IN MS

2A, 2B). In experiments performed as in Fig. 3D, with 100 mg/l of observed upregulation of IL22 transcription in CD4 T cells doxycycline, the transgenic rats have significantly smaller lymph after 24 h of stimulation (Fig. 4B). IL22RA1 was detected ex nodes 7 d after immunization (Supplemental Fig. 4C, 4D), con- vivo in both subsets, but no significant change was induced sistent with a facilitating role of IL-22BP on immune cell priming/ after 24 h of polyclonal stimulation (Fig. 4C). To test if addi- expansion. When assessing T cell polarization in draining lymph tional IL-22 stimulation has an effect on IFN-g production, we nodes 7 d after MOG immunization, we observed a significant activated CD4 or CD8 T cells for 3 d and stimulated them si- decrease in Ag-specific IFN-g+ T cells (Fig. 4A). This suggests multaneously with recombinant human IL-22. A statistically that IL-22 acts to dampen the immune response, with a prominent significant difference was seen with higher IFN-g production in effect on Th1 polarization. We set out to investigate this in human CD4 T cells that had not been activated in the presence of IL-22 T cells and performed polyclonal stimulation of human peripheral (Fig. 4D). A similar trend was observed in cultures of CD8 blood CD4 or CD8 T cells with Abs for CD3 and CD28. We T cells. Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. IL-22BP promotes the Th1 response. (A) A total of 100 mg/l of doxycycline was added to the drinking water 4 d prior to MOG immunization, leading to knockdown of Il22ra expression during immune cell priming in rats carrying the transgene. Seven days after immunization, lymph node T cells were restimulated with MOG in vitro and phenotyped by flow cytometry. Results are mean 6 SEM, n = 6 per group. Significance testing was performed using two-tailed Mann–Whitney U test, and p values have been Bonferroni corrected for four tests. Data are representative of two independent successive experiments. (B and C) CD4+ and CD8+ human healthy donor PBMCs were cultured separately and activated with beads coated with Abs for CD3 and CD28 (n = 6 per group). Cells were harvested after 24 h, and expression of IL22 and IL22RA1 was assessed by qPCR and compared with expression levels ex vivo (mean 6 SEM). Significance testing was performed using two-tailed Wilcoxon matched-pairs signed-rank test, and p values have been Bonferroni corrected for two tests. Data represent two independent successive experiments. (D) The same type of samples were cultured for 72 h with the addition of recombinant human IL-22 (2 mg/ml) followed by assessment of IFN-g production by flow cytometry (n = 8 per group). Stimulated and unstimulated samples from the same donor are connected by a black line. Significance testing was performed using two-tailed paired Student t test (D’Agostino and Pearson normality test, p = 0.91 and 0.66, respectively), and p values have been Bonferroni corrected for two tests. Data are compiled from two successive experiments. (E) Human PBMCs were cultured for 3.5 h with cell medium only or with the addition of LPS (10 mg/ml) followed by assessment of IFNG expression by qPCR. Results are the fold change of IFNG expression in LPS-stimulated wells compared with control wells (mean 6 SEM). Carriers of the risk G genotype of rs17066096 (n = 36) are compared with noncarriers (n = 46). Significance testing was performed using Mann– Whitney U test (D’Agostino and Pearson normality test, p , 0.0001 for both genotypes). The Journal of Immunology 9

Our data suggest that carriers of the risk genotype rs17066096G alone had no effect on EAE score, which has been reported pre- have higher expression of IL-22 antagonist IL-22BP. Furthermore, viously by others (27). There was no difference in day of disease in this study, we show rat and human data implying that IL-22 has onset when comparing the four strains. Infiltrating immune cells the potential of suppressing the Th1 signature cytokine IFN-g. were isolated from whole spinal cord of perfused mice on day 29 Consequently, we hypothesized that carriers of rs17066096G of EAE and characterized by flow cytometry. Il22ra22/2 mice had would, via decreased availability of IL-22, exhibit a higher ca- fewer infiltrating T cells compared with wild-type mice as well as pacity for IFN-g production compared with noncarriers. To in- the double knockout mice (Fig. 5C). Interestingly, Il222/2 mice vestigate this, we used samples available to us from cultures of had more infiltration of neutrophils and a trend toward more PBMCs from 84 patients with MS or other neurologic diseases T cells and inflammatory monocytes, compared with wild-type with known rs17066096 genotype. These cells had been stimu- mice. lated for 3.5 h in medium only or with the addition of LPS, thus approximating physiological activation of memory T cells via Discussion innate immune cells. We determined the levels of IFNG gene We were first, to our knowledge, to link IL-22BP to neuro- expression in these samples and observed significantly higher inflammation in a study of rat and human natural genetic variation levels in carriers of the risk genotype (Fig. 4E). This is consistent in this locus, demonstrating effects on EAE severity as well as MS with the role of IFN-g as an activator of innate immune cells, the susceptibility and severity (17). The protective genetic variant in effector arm of the Th1 immune response, and also a documented the rat strain used in that study (17) conferred lower expression of pathogenic role of IFN-g in MS (26). IL-22BP, making it the candidate gene for the effects seen in EAE. We then went on to pinpoint the effect on EAE to the actual gene Downloaded from IL-22BP deficiency reduces severity of mouse EAE in an using IL-22BP–deleted mice (18), which have less severe disease. IL-22–dependent manner We have since then shown, in three large-scale studies, that the G Next, we asked if the disease-driving effect of IL-22BP on EAE that allele of SNP rs17066096, located 14 kb downstream of the gene we observed in this study in rats, and have previously reported in for IL-22BP, is associated with higher MS susceptibility (3–5). mice (18), is mediated by IL-22. To investigate this, we performed The combined p value of 9.26 3 10236 makes this one of the

EAE experiments with mice in which either Il22 or Il22ra2 strongest MS-associated non-HLA variants (5). It was not known http://www.jimmunol.org/ was deleted or in which both genes were deleted. We observed how this increased risk is mediated, but based on the data that we less-severe disease in Il22ra22/2 mice compared with wild-type have published on mice and rats, we hypothesized that the MS- mice (Fig. 5A). Interestingly, heterozygous deletion of Il22ra2 associated G allele would be associated with higher expression of was sufﬁcient to achieve the full protective effect. However, the IL-22BP, thereby mediating the pathogenic effect. protective effect of Il22ra2 deletion was completely lost when the Overall, there are very few examples of uncovered mechanisms Il22 gene was also deleted (Fig. 5B). The absence of the Il22 gene behind genetic associations to complex diseases in the literature, by guest on September 24, 2021

FIGURE 5. Il22ra2 deletion reduces severity of EAE in an IL-22–dependent manner. (A) EAE in wild-type (WT), Il22ra2+/2, and Il22ra22/2 mice. (B) EAE in WT, Il222/2, Il22ra2/2, and Il22ra22/2 Il222/2 (double knockout [KO]) mice. Results are compiled from four successive experiments with similar results (mean 6 SEM, n = 13–24). Significance testing was performed using Kruskal–Wallis test applying Dunn’s post hoc test. (C) Spinal cord–infiltrating cells were characterized by flow cytometry, and basic populations were defined as CD45high CD11b2 CD3+ T cells, CD45+ CD11b+ Ly6G2 inflammatory monocytes, and CD45+ CD11b+ Ly6G+ neutrophils. Results are compiled from two successive experiments with similar results (mean 6 SEM; WT, n = 11; Il222/2, n =7;Il22ra2/2, n = 12; double KO, n = 6). Significance testing was done using one-way ANOVA, applying Tukey post hoc test, in which *p , 0.05, **p , 0.01, ***p , 0.001. 10 IL-22BP PROMOTES THE DISEASE PROCESS IN MS especially when considering noncoding variants. However, several activation. Our data suggest that the latter is at least plausible, successful studies have, like we have done in this study, used considering the anti-inflammatory effect on rat and human T cells. samples from healthy individuals (28–34). In the current study, we Further support for this interpretation can be found in a study of first defined an in vitro system in which IL-22BP gene expression experimental autoimmune uveitis, in which administration of IL-22 can be studied, necessitated by the fact that mRNA levels are not on day 4 and 8 after immunization results in milder disease (36). readily detected in circulating cells. Just like rodent tissue DCs They report that in vitro stimulation of T cells from IL-22–treated (18), these human in vitro differentiated monocytes produced high mice results in decreased IL-17 and IFN-g secretion as well as an levels of IL-22BP, providing a system to study the role of genetic increased proportion of Foxp3+ T cells. Results that are essentially variation. This approach has also been used successfully by others similar to what we show in this study but using a different ex- (30). In fact, emerging data suggest that noncoding variants often perimental intervention, disease model, and species. Moreover, require specific context to exert their effect, and this has recently they also detect upregulation of IL-22R in lymphoid organs after also been suggested for autoimmune disease risk genes, including immunization but localize it to APCs rather than T cells. However, MS (20, 35). When studying IL-22BP expression using this ap- the method of isolation in that report was adherence to nylon wool proach, we observed context-dependent differences relative to columns, which is known to alter the activation state and marker rs17066096 genotype, consistent with an eQTL. Furthermore, expression of T cells (37). CSF IL-22BP levels were higher in MS patients with more severe In this study, we show IL-22BP expression in the CNS. The neuroinflammation. The pathogenic effect of IL-22BP in neuro- major CNS cell types were sorted from untreated neonatal wild- inflammation was demonstrated in mouse and rat EAE, which type mice, and IL-22BP expression was present and restricted to correlated with a reduction in IFN-g production from draining CD11b+ cells. Moreover, IL-22R was detected in mouse brain Downloaded from lymph node encephalitogenic T cells. Moreover, rat lymph node samples enriched for O4+ oligodendroglia and to a lesser extent in cells had upregulated the membrane-bound receptor 7 d after astrocytes. Two previous studies have addressed IL-22 signaling EAE induction. Translating the findings back to human cells, we on glial cells, arriving at somewhat contradictory conclusions. demonstrate the ability of IL-22 to limit T cell IFN-g production Human astrocytes cultured under stress are protected by IL-22, and provide further evidence for this effect as well as the eQTL in whereas mouse oligodendrocytes are more likely to go into apo-

an experiment using cells from a large number of patients with MS ptosis under similar conditions (25, 38). In our assays, IL-22 http://www.jimmunol.org/ and other neurologic diseases. stimulation of mouse and rat glial cells had no effect, and In an early report, IL-22–deleted mice were shown to have knocking down IL-22BP after immune cell activation had no an unaltered clinical and histopathological presentation of EAE strong effect on disease course of EAE in rats. IL-22BP expressed compared with wild-type mice (27). In contrast, we show that in the periphery is thus likely to have a more decisive role in the IL-22BP–deleted mice have less severe EAE compared with wild- context of EAE but not necessarily in MS. type mice. One can speculate that the presence of IL-22BP, and its In summary, although the knowledge of the genetic architecture efficient IL-22 antagonism, masks the potential of IL-22 to sig- of MS susceptibility has expanded immensely in the last few years, nificantly influence EAE disease course when comparing with real clinical benefit from these efforts will require functional an- IL-22–deleted mice. Another possible explanation is that the ef- notations of the associated sequence variants. In this study, we have by guest on September 24, 2021 fect of IL-22BP is mediated independently of IL-22. In this study, focused on one of the strongest MS-associated non-HLAvariants as we addressed this by conducting a series of EAE experiments with well as the nearby localized gene for IL-22BP. We have presented wild-type, IL-22–deleted, and IL-22BP–deleted mice as well as evidence for a protective role of IL-22 in the context of MS, thus mice in which both genes are deleted. We could conclude that the advancing our understanding of this enigmatic disease and sug- reduced severity of disease in IL-22BP–deleted mice is an effect gesting IL-22BP as a novel MS drug target. of disinhibited IL-22 signaling. Moreover, like the previous study of EAE in IL-22–deleted mice (27), we saw no effect on EAE score, but when analyzing the infiltrating cells, we detected more Disclosures neutrophils and a trend toward more T cells. This further supports The authors have no financial conflicts of interest. a protective role for IL-22 in neuroinflammation, but it also shows that this effect is to a large degree restrained in wild-type mice because of the presence of IL-22BP. Another interesting aspect of References 1. Olsson, T., L. F. Barcellos, and L. Alfredsson. 2017. Interactions between ge- the mouse EAE experiments is that heterozygous deletion of the netic, lifestyle and environmental risk factors for multiple sclerosis. Nat. Rev. IL-22BP gene is sufficient to achieve the full protective effect, Neurol. 13: 25–36. analogous with the subtle knockdown of IL-22BP in the rat EAE 2. Thompson, A. J., S. E. Baranzini, J. Geurts, B. Hemmer, and O. Ciccarelli. 2018. Multiple sclerosis. Lancet 391: 1622–1636. experiments, which serves as indirect evidence that the moderate- 3. Sawcer, S., G. Hellenthal, M. Pirinen, C. C. Spencer, N. A. Patsopoulos, sized modulation of human IL-22BP expression by the risk variant L. Moutsianas, A. Dilthey, Z. Su, C. Freeman, S. E. Hunt, et al; Wellcome Trust is clinically relevant. Case Control Consortium 2. 2011. Genetic risk and a primary role for cell- mediated immune mechanisms in multiple sclerosis. Nature 476: 214–219. The inverse patterns of IL-22 and IL-22BP expression in the 4. Beecham, A. H., N. A. Patsopoulos, D. K. Xifara, M. F. Davis, A. Kemppinen, lymph node that we observed during the first days after EAE in- C. Cotsapas, T. S. Shah, C. Spencer, D. Booth, A. Goris, et al; International IBD Genetics Consortium (IIBDGC). 2013. Analysis of immune-related loci identifies duction are consistent with the existence of common upstream 48 new susceptibility variants for multiple sclerosis. Nat. Genet. 45: 1353–1360. signaling events regulating transcription in a manner that facilitates 5. International Multiple Sclerosis Genetics Consortium. 2019. The Multiple IL-22 signaling, that is, as IL-22 increases, the homeostatic ex- Sclerosis Genomic Map: role of peripheral immune cells and resident microglia in susceptibility. Available at: https://www.biorxiv.org/content/early/2017/07/13/ pression of antagonist molecule IL-22BP is dampened. These 143933. Accessed: July 1, 2019. events could be interpreted as a physiological upregulation of IL-22 6. Couturier, N., F. Bucciarelli, R. N. Nurtdinov, M. Debouverie, C. Lebrun-Frenay, signaling as a consequence of tissue damage from the s.c. injection G. Defer, T. Moreau, C. Confavreux, S. Vukusic, I. Cournu-Rebeix, et al. 2011. Tyrosine kinase 2 variant influences T lymphocyte polarization and multiple of Ag emulsified with adjuvant, with a concomitant decrease in sclerosis susceptibility. Brain 134: 693–703. IL-22BP to facilitate the tissue-regenerative actions of the cytokine. 7. Gregory, A. P., C. A. Dendrou, K. E. Attfield, A. Haghikia, D. K. Xifara, F. Butter, G. Poschmann, G. Kaur, L. Lambert, O. A. Leach, et al. 2012. TNF Alternatively, IL-22 is induced as a physiological feedback inhi- receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple scle- bition of the events during immune cell priming to limit excessive rosis. Nature 488: 508–511. The Journal of Immunology 11

8. Hartmann, F. J., M. Khademi, J. Aram, S. Ammann, I. Kockum, 23. Storch, M. K., A. Stefferl, U. Brehm, R. Weissert, E. Wallstro¨m, C. Constantinescu, B. Gran, F. Piehl, T. Olsson, L. Codarri, and B. Becher. 2014. M. Kerschensteiner, T. Olsson, C. Linington, and H. Lassmann. 1998. Autoim- Multiple sclerosis-associated IL2RA polymorphism controls GM-CSF produc- munity to myelin oligodendrocyte glycoprotein in rats mimics the spectrum of tion in human TH cells. Nat. Commun. 5: 5056. multiple sclerosis pathology. Brain Pathol. 8: 681–694. 9. Gregory, S. G., S. Schmidt, P. Seth, J. R. Oksenberg, J. Hart, A. Prokop, 24. Dudakov, J. A., A. M. Hanash, and M. R. M. van den Brink. 2015. Interleukin- S. J. Caillier, M. Ban, A. Goris, L. F. Barcellos, et al; Multiple Sclerosis Genetics 22: immunobiology and pathology. Annu. Rev. Immunol. 33: 747–785. Group. 2007. Interleukin 7 receptor a chain (IL7R) shows allelic and functional 25. Perriard, G., A. Mathias, L. Enz, M. Canales, M. Schluep, M. Gentner, association with multiple sclerosis. Nat. Genet. 39: 1083–1091. N. Schaeren-Wiemers, and R. A. Du Pasquier. 2015. Interleukin-22 is increased 10. Dendrou, C. A., A. Cortes, L. Shipman, H. G. Evans, K. E. Attfield, L. Jostins, in multiple sclerosis patients and targets astrocytes. J. Neuroinflammation 12: T. Barber, G. Kaur, S. B. Kuttikkatte, O. A. Leach, et al. 2016. Resolving TYK2 119. locus genotype-to-phenotype differences in autoimmunity. Sci. Transl. Med. 8: 26. Panitch, H. S., R. L. Hirsch, A. S. Haley, and K. P. Johnson. 1987. Exacerbations 363ra149. of multiple sclerosis in patients treated with gamma interferon. Lancet 1: 893– 11. Dumoutier, L., D. Lejeune, D. Colau, and J. C. Renauld. 2001. Cloning and 895. characterization of IL-22 binding protein, a natural antagonist of IL-10-related 27. Kreymborg, K., R. Etzensperger, L. Dumoutier, S. Haak, A. Rebollo, T. Buch, T cell-derived inducible factor/IL-22. J. Immunol. 166: 7090–7095. F. L. Heppner, J.-C. C. Renauld, and B. Becher. 2007. IL-22 is expressed by 12. Wu, P. W., J. Li, S. R. Kodangattil, D. P. Luxenberg, F. Bennett, M. Martino, Th17 cells in an IL-23-dependent fashion, but not required for the development M. Collins, K. Dunussi-Joannopoulos, D. S. Gill, N. M. Wolfman, and of autoimmune encephalomyelitis. J. Immunol. 179: 8098–8104. L. A. Fouser. 2008. IL-22R, IL-10R2, and IL-22BP binding sites are topologi- 28. Dendrou, C. A., V. Plagnol, E. Fung, J. H. M. M. Yang, K. Downes, J. D. Cooper, cally juxtaposed on adjacent and overlapping surfaces of IL-22. J. Mol. Biol. S. Nutland, G. Coleman, M. Himsworth, M. Hardy, et al. 2009. Cell-specific 382: 1168–1183. protein phenotypes for the autoimmune locus IL2RA using a genotype- 13. Wolk, K., S. Kunz, E. Witte, M. Friedrich, K. Asadullah, and R. Sabat. 2004. selectable human bioresource. Nat. Genet. 41: 1011–1015. IL-22 increases the innate immunity of tissues. Immunity 21: 241–254. 29. Pidasheva, S., S. Trifari, A. Phillips, J. A. Hackney, Y. Ma, A. Smith, S. J. Sohn, 14. Sabat, R., W. Ouyang, and K. Wolk. 2014. Therapeutic opportunities of the H. Spits, R. D. Little, T. W. Behrens, et al. 2011. Functional studies on the IBD IL-22-IL-22R1 system. Nat. Rev. Drug Discov. 13: 21–38. susceptibility gene IL23R implicate reduced receptor function in the protective 15. Lindahl, H., E. Martini, S. Brauner, P. Nikamo, I. Gallais Serezal, genetic variant R381Q. PLoS One 6: e25038. A. O. Guerreiro-Cacais, M. Jagodic, L. Eidsmo, M. Stahle,˚ and T. Olsson. 2017. 30. Kim, S. J., P. K. Gregersen, and B. Diamond. 2013. Regulation of dendritic cell Downloaded from IL-22 binding protein regulates murine skin inflammation. Exp. Dermatol. 26: activation by microRNA let-7c and BLIMP1. J. Clin. Invest. 123: 823–833. 444–446. 31. Hu, X., H. Kim, T. Raj, P. J. Brennan, G. Trynka, N. Teslovich, K. Slowikowski, 16. Huber, S., N. Gagliani, L. A. Zenewicz, F. J. Huber, L. Bosurgi, B. Hu, M. Hedl, W.-M. Chen, S. Onengut, C. Baecher-Allan, et al. 2014. Regulation of gene W. Zhang, W. O’Connor, Jr., A. J. Murphy, et al. 2012. IL-22BP is regulated by expression in autoimmune disease loci and the genetic basis of proliferation in the inflammasome and modulates tumorigenesis in the intestine. Nature 491: CD4+ effector memory T cells. PLoS Genet. 10: e1004404. 259–263. 32. Chang, H.-H., N. Dwivedi, A. P. Nicholas, and I.-C. Ho. 2015. The W620 17. Beyeen, A. D., M. Z. Adzemovic, J. Ockinger, P. Stridh, K. Becanovic, polymorphism in PTPN22 disrupts its interaction with peptidylarginine deimi-

H. Laaksonen, H. Lassmann, R. A. Harris, J. Hillert, L. Alfredsson, et al. 2010. nase type 4 and enhances citrullination and NETosis. Arthritis Rheumatol. 67: http://www.jimmunol.org/ IL-22RA2 associates with multiple sclerosis and macrophage effector mecha- 2323–2334. nisms in experimental neuroinflammation. J. Immunol. 185: 6883–6890. 33. Simpfendorfer, K. R., B. E. Armstead, A. Shih, W. Li, M. Curran, N. Manjarrez- 18. Laaksonen, H., A. O. Guerreiro-Cacais, M. Z. Adzemovic, R. Parsa, Ordunõ, A. T. Lee, B. Diamond, and P. K. Gregersen. 2015. Autoimmune disease- M. Zeitelhofer, M. Jagodic, and T. Olsson. 2014. The multiple sclerosis risk gene associated haplotypes of BLK exhibit lowered thresholds for B cell activation and IL22RA2 contributes to a more severe murine autoimmune neuroinflammation. expansion of Ig class-switched B cells. Arthritis Rheumatol. 67: 2866–2876. Genes Immun. 15: 457–465. 34. Di Meglio, P., A. Di Cesare, U. Laggner, C.-C. Chu, L. Napolitano, F. Villanova, 19. Chen, Y., B. Stevens, J. Chang, J. Milbrandt, B. A. Barres, and J. W. Hell. 2008. I. Tosi, F. Capon, R. C. Trembath, K. Peris, and F. O. Nestle. 2011. The IL23R NS21: re-defined and modified supplement B27 for neuronal cultures. J. Neu- R381Q gene variant protects against immune-mediated diseases by impairing IL- rosci. Methods 171: 239–247. 23-induced Th17 effector response in humans. PLoS One 6: e17160. 20. Alasoo, K., J. Rodrigues, S. Mukhopadhyay, A. J. Knights, A. L. Mann, 35. Shooshtari, P., H. Huang, and C. Cotsapas. 2017. Integrative genetic and epi- K. Kundu, C. Hale, G. Dougan, and D. J. Gaffney; HIPSCI Consortium. 2018. genetic analysis uncovers regulatory mechanisms of autoimmune disease. Am. J.

Shared genetic effects on chromatin and gene expression indicate a role for Hum. Genet. 101: 75–86. by guest on September 24, 2021 enhancer priming in immune response. Nat. Genet. 50: 424–431. 36. Ke, Y., D. Sun, G. Jiang, H. J. Kaplan, and H. Shao. 2011. IL-22-induced reg- 21. Martin, J. C. J., G. Be´riou, M. Heslan, C. Chauvin, L. Utriainen, A. Aumeunier, ulatory CD11b+ APCs suppress experimental autoimmune uveitis. J. Immunol. C. L. Scott, A. Mowat, V. Cerovic, S. A. Houston, et al. 2014. Interleukin-22 187: 2130–2139. binding protein (IL-22BP) is constitutively expressed by a subset of conventional 37. Wohler, J. E., and S. R. Barnum. 2009. Nylon wool puriﬁcation alters the acti- dendritic cells and is strongly induced by retinoic acid. Mucosal Immunol. 7: vation of T cells. Mol. Immunol. 46: 1007–1010. 101–113. 38. Zhen, J., J. Yuan, Y. Fu, R. Zhu, M. Wang, H. Chang, Y. Zhao, D. Wang, and 22. Weissert, R., E. Wallstro¨m, M. K. Storch, A. Stefferl, J. Lorentzen, H. Lassmann, Z. Lu. 2017. IL-22 promotes Fas expression in oligodendrocytes and inhibits C. Linington, and T. Olsson. 1998. MHC haplotype-dependent regulation of FOXP3 expression in T cells by activating the NF-kB pathway in multiple MOG-induced EAE in rats. J. Clin. Invest. 102: 1265–1273. sclerosis. Mol. Immunol. 82: 84–93.

SUPPLEMENTAL FIGURE 1. MS risk locus rs17066096. Idiogram of chromosome 6 showing the location of the MS risk locus defined by the marker rs17066096. The graph displays a magnification of the locus showing the position in base pairs, nearest protein coding genes with splice variants, H3K4Me1 (often found near regulatory elements), H3K4Me3 (often found near promotors), H3K27Ac (often found near active regulatory elements, DNase I hypersensitivity clusters, and transcription factor ChIP-seq clusters, all from the ENCODE project. Image is adapted from the UCSC Genome Browser (hg19). Linkage disequilibrium (LD) plot showing R2 values represented as a heat map in which red indicates high LD, based on 1000 Genomes phase 3 CEU population. LD plot is adapted from the Ensembl Genome Browser.

SUPPLEMENTAL FIGURE 2. AM580-IL22RA2 dose-response curve and CSF IL-22BP vs age or EDSS. (A) Human peripheral blood monocytes were differentiated with GM-CSF and IL- 4 for 6 days. Increasing amounts of AM580 or the same volume of vehicle (DMSO) was added from the start of the differentiation. (B) Human IL-22BP, determined by ELISA, in CSF from MS patients plotted against age and Expanded Disability Status Scale (EDSS). Spearman’s rank correlation coefficient and two-tailed P-value are reported in the figure.

3 SUPPLEMENTAL FIGURE 3. The components of the IL-22-system are expressed in distinct cell populations in the mouse CNS. (A) Brains from naïve p2 neonatal C57BL/6 mice (n = 5) or adult mice taken day 29 of an EAE experiment (n = 2) were dissociated and the cells were enriched using magnetic beads for astrocytes (ACSA-1 and ACSA-2 combined), oligodendroglia (O4), microglia (CD11b), and neurons (CD171) followed by quantification of Il22, Il22ra2, and Il22ra1 mRNA. (B) Expression of cell-type specific genes for the purpose of assessing sorting purity. (C) Rat oligodendrocyte precursor cells were differentiated with or without the addition of recombinant rat IL-22 in the medium. After 6 days, differentiation was assessed by quantifying cells expressing myelin basic protein (MBP) as well as overall cell number (nuclei). Presence of astrocytes could also be assessed by measuring the area with positive staining for glial fibrillary acidic protein (GFAP). Results are reported as group means +/- SEM. Representative stainings are shown. (D) Mouse astrocytes were isolated and cultured with a panel of conditions that induce apoptosis with or without the addition of recombinant mouse IL-22. The potential of IL-22 to influence apoptosis was examined by Annexin V-staining that was quantified using flow cytometry (n = 2). The histograms show representative Annexin V staining from samples without IL-22 stimulation. Results are reported as group means +/- SEM.

SUPPLEMENTAL FIGURE 4. Doxycycline treatment titration for rat strain with doxycycline-induced gene-specific knockdown. (A) Expression of Il22ra2 in inguinal lymph nodes of rats in relation to concentration of doxycycline in the drinking water and duration of administration. Values are relative to baseline expression. Each data point represents one unique rat. (B) Expression of Il22ra2 in tissue samples from naïve wild type DA rats treated with 100 mg/l of doxycycline for 10 days. The values in Fig. 3B are expressed as percentages of these. Wild type (white) and rats carrying the transgene (black) were treated with 100 mg/l of doxycycline in the drinking water for 10 days and were either (C) immunized with MOG after day 3 or (D) kept without immunization. Results are reported as group means +/- SEM and P-values have been calculated using two-tailed unpaired Student’s t-test.