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 J Immunol published online 10 July 2019 Downloaded from http://www.jimmunol.org/content/early/2019/07/09/jimmun ol.1900400 Supplementary http://www.jimmunol.org/content/suppl/2019/07/10/jimmunol.190040 http://www.jimmunol.org/ Material 0.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 path- ogenic 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. Linde´n, 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 anal- ysis. H.L. wrote the manuscript. H.L., A.O.G.-C., S.K.B., M. Linnerbauer, M. Linde´n, 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, ex- perimental 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.