532700
research-article2014
MSJ0010.1177/1352458514532700Multiple Sclerosis JournalW Lundström, C Hermanrud
MULTIPLE SCLEROSIS MSJ JOURNAL
Research Paper
Interferon beta treatment of multiple sclerosis increases serum interleukin-7 Wangko Lundström, Christina Hermanrud, Maria Sjöstrand, Susanna Brauner, Marie WahrenHerlenius, Tomas Olsson, Virginija Karrenbauer, Jan Hillert and Anna Fogdell-Hahn
Multiple Sclerosis Journal 2014, Vol. 20(13) 1727–1736 DOI: 10.1177/ 1352458514532700 © The Author(s), 2014. Reprints and permissions: http://www.sagepub.co.uk/ journalsPermissions.nav
Abstract Background: Interleukin-7 (IL-7) is a non-redundant cytokine for T-cell development and survival. The IL-7 signaling pathway has been genetically and functionally associated with several autoimmune diseases including multiple sclerosis (MS). Objective: The objective of this paper is to elucidate the effect of the widely used immunomodulatory MS therapy interferon beta (IFNβ) on IL-7 homeostasis. Methods: Swedish MS patients were screened for IL-7 concentration in serum and blood cell counts. IL-7 receptor alpha chain (IL-7Rα) expression was determined by semi-quantitative real-time polymerase chain reaction (PCR) and flow cytometry. Results: IFNβ treatment led to significantly increased serum IL-7 levels (mean: 17 pg/ml) compared with healthy controls (mean: 7.6 pg/ml) and natalizumab-treated patients (mean: 5.3 pg/ml). In vitro and in vivo, peripheral blood leukocytes showed decreased IL-7Rα expression and IL-7 consumption upon IFNβ exposure, suggesting that their IL-7 responsiveness is impaired during treatment. Conclusions: MS patients undergoing IFNβ treatment have increased serum IL-7 levels and decreased IL-7 consumption. Given IL-7’s important role in T-cell immunity, this relationship may be highly relevant for IFNβ’s treatment efficacy.
Keywords: Multiple sclerosis, immunology, interferon beta, natalizumab Date received: 16 September 2013; revised: 10 March 2014; accepted: 30 March 2014
Introduction The exact cause of multiple sclerosis (MS) remains unknown, but thanks to twin- and migration studies it is clear that both genetic and environmental factors contribute to determine disease risk.1 Human leukocyte antigen (HLA) was the first genetic region associated with MS susceptibility, and HLA-DRB1*15 remains the strongest known genetic predisposition.2–5 Over the last few years the list of MS susceptibility linked genes has grown rapidly with the introduction of genomewide association studies (GWAS). Although explaining a substantial part of MS causality, the genotypes that increase susceptibility seem to have no impact on disease severity, suggesting separate mechanisms regulating disease onset versus progress.6–8 The first confirmed genetic association to MS susceptibility outside the HLA region was in the interleukin-7 receptor gene (IL7R).9–11
IL7R encodes the IL-7 receptor alpha chain (IL-7Rα or CD127), which forms the IL-7 receptor when in complex with the common gamma chain (CD132), and the thymic stromal lymphopoietin receptor (TSLPR) when in complex with the TSLPR chain. Apart from the IL7R association, a recent multicenter GWAS confirmed the IL7 gene as a genetic modulator of MS susceptibility.5 Interferon beta (IFNβ) was the first approved drug for treatment of relapsing–remitting MS and has been used as a first-line disease-modifying agent over the past two decades. The exact mechanism by which IFNβ modulates MS progress remains unknown, but immune modulation is widely accepted as, at least part of, the explanation.12,13 Treatment with recombinant human IFNβ has showed both decreased relapse rate and reduced size
Correspondence to: Anna Fogdell-Hahn Center for Molecular Medicine, Karolinska Institutet, SE-17176 Stockholm, Sweden. [email protected] Wangko Lundström Christina Hermanrud Tomas Olsson Virginija Karrenbauer Jan Hillert Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Maria Sjöstrand Susanna Brauner Marie Wahren-Herlenius Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Sweden
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Multiple Sclerosis Journal 20(13) of brain lesions,14 hence early treatment start could potentially reduce disability.15 Three types of IFNβ preparations are currently available for treatment of MS: intramuscular (IM) IFNβ-1a (Avonex, Biogen Idec, Weston, MA, USA), subcutaneous (SC) IFNβ-1a (Rebif 22/44 µg, Serono, Geneva, Switzerland) and SC IFNβ-1b (Betaferon/Extavia, Bayer/Novartis, Basel, Switzerland). Up to 47 percent of patients being treated with IFNβ will develop neutralizing antibodies (NAbs), which can abrogate the bioactivity of the drug.16 Presence of NAbs is therefore an important factor to consider when determining proper therapy for MS patients. Over the past few years, IL-7 and its receptor have been shown to influence autoimmunity in various diseases and animal models.17 In order to optimize MS treatment with IFNβ, it is important to understand its impact on endogenous immune processes. In this work we present in vitro and in vivo investigations elucidating the interplay between IFNβ administration and IL-7 biology. Materials and methods Study group Serum samples from 184 Swedish MS patients receiving IFNβ treatment and tested for NAbs at the NAb routine laboratory at Karolinska Institutet were included in this study. Patients were treated with different IFNβ preparations, either IFNβ-1a (Avonex, n = 73; Rebif 22 μg, n = 10; Rebif 44 μg, n = 58), or IFNβ-1b (Betaferon, n = 43). Serum samples were collected one to seven days post-IFNβ injection. Patients with or without NAbs to IFNβ were included in the study. The control cohorts were composed of serum samples retrieved from six healthy volunteers and 21 natalizumab-treated MS patients. Serum samples from healthy volunteers were obtained from the Karolinska Hospital blood bank and MS patients on natalizumab therapy from the NAb routine laboratory at Karolinska Institutet, and all were NAb negative. All patients on natalizumab therapy had previously been treated with IFNβ and been on natalizumab treatment for at least six months. For IL7R gene expression analysis, serial blood samples were collected from eight MS patients, who were treatment naïve before study entry. Samples were taken from each patient before IM IFNβ-1a treatment onset, approximately 18 hours after the first injection, and three to 13 weeks post-treatment start-up and 12–18 hours after last injection.
Data from the Immunomodulation and MS Epidemiology (IMSE) cohort at Karolinska University Hospital, Huddinge was retrieved for MS patients treated with IFNβ or natalizumab. Leukocyte, lymphocyte and monocyte populations as well as whole blood counts were quantified by flow cytometry at a clinical laboratory using the XE-5000 system (Sysmex co, Bremerhaven, DE). The values of leukocyte, lymphocyte, and monocyte numbers were grouped as follows: before IFNβ treatment start (n = 16), during IFNβ treatment (NAb– n = 25; NAb+ n = 12), and during natalizumab treatment (n = 18). For a summary of patient demographics, see Supplementary Table 1. NAb analysis MS patients were screened for the presence of antiIFNβ antibody neutralizing activity using the myxovirus resistance protein A (MxA) gene expression bioassay (MGA).18,19 Patient serum was incubated with A549 cells, and the ability of patient sera to neutralize the added recombinant IFNβ-1a (Avonex®, Biogen Idec Inc, Cambridge, MA, USA) was quantified by 7500 Real-Time polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA, USA). Antibody neutralizing activity titers were adjusted according to the Kawade formula20 using Softmax Pro software (Sunnyvale, CA, USA). The NAb titers were expressed as tenfold reduction units per milliliter (TRU/ml) and the patients were classified according to the following categories: negative (1280 TRU/ml). Cell isolation from buffy coat and cell cultures Peripheral blood mononuclear cells (PBMC) from whole blood buffy coats from healthy blood donors (Karolinska Hospital blood bank) were isolated by Ficoll-Hypaque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. PBMCs were counted using a hemocytometer and resuspended to a concentration of 1 × 107 cells/ml and cryopreserved until used. The viability of the cells was determined by trypan blue. A total of 1 × 106 cells/ml of cryopreserved PBMCs were cultured in medium consisting of Roswell Park Memorial Institute (RPMI) 1640 medium (GIBCO, Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal calf serum (Thermo Fisher, Waltham, MA, USA), and 1% penicillinstreptomycin (Life Technologies) at 37°C in 5% CO2. Flow cytometry PBMCs were cultured at 106 cells/ml in 400 µl medium per well of a 24-well plate (NUNC) with
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W Lundström, C Hermanrud et al. recombinant IFNβ-1a (Avonex®, Biogen Idec Inc, Cambridge, MA, USA) at various concentrations (1000, 100, 10, 1, 0.1, 0 IU/ml). After one, three, five, and seven days of incubation, cells were stained with CD8-PB (Life Technologies), CD3APC, CD4-PerCP, CD14-fluorescein isothiocyanate (FITC) (BD Biosciences, Franklin Lakes, NJ, USA), CD19-PE and CD127-PE-Cy7 (BioLegend, San Diego, CA, USA) at 4°C for 30 minutes. The samples were acquired on a CyAn Dako flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed by FlowJo software (Tree Star, Ashland, OR, USA) and GraphPad Prism (La Jolla, CA, USA). Data are representative of three independent experiments. IL-7 receptor-mediated IL-7 consumption PBMCs from healthy volunteers were cultured in 200 µl medium per well in a 96-well plate (NUNC) with recombinant human IL-7 (Roskilde, Denmark) at various concentrations (1000, 100, 10, 0 pg/ml) with or without recombinant IFNβ-1a (Avonex®, Biogen Idec Inc) at 1000 IU/ml. After four, 21, 45, 72 and 216 hours of incubation, cells were counted and IL-7 levels in the culture medium were quantified by a commercially available enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (HS IL-7 ELISA kit, R&D Systems, Minneapolis, MN, USA). Data are representative of three independent experiments. Real-time quantitative PCR Total RNA from CD3+ fluorescence-activated cell sorted (FACS) T-cells was isolated using Trizol (Life Technologies) according to the manufacturer’s protocol. To enhance RNA precipitation 50 µg of glycogen (Thermo Fisher) was added. The RNA concentration was determined by NanoDrop (Thermo Fisher). RNA was reverse transcribed to cDNA by iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Expression level of full length membrane-bound IL-7Rα (presence of exon 6) and the soluble form of IL-7Rα (absence of exon 6) was measured using C1000 Thermal Cycler (Bio-Rad) commercially available primer-probe solutions (Life Technologies; full-length IL-7Rα: Hs00904814_m1, sIL-7Rα: Hs00902337_m1, and HPRT1: Hs01003267_m1). Expression levels were normalized to HPRT1. Expression values were calculated using the 2ΔΔCt formula where Ct values were the mean of technical duplicates. Statistical analyses Statistical calculations were performed using Prism software (GraphPad Inc). p values were calculated by
unpaired, two-sided t tests and statistical significance was defined as a p value < 0.05. Ethical considerations Informed consent for all parts of the study were obtained from all patients and studies were approved by the regional ethics board in Stockholm. Results IFNβ treatment leads to elevated serum IL-7 levels in MS patients To assess the potential impact of IFNβ treatment on IL-7 homeostasis, we first determined serum IL-7 levels in 205 MS patients and six healthy blood donors by ELISA. Patients receiving IFNβ treatment (n = 184) had significantly increased IL-7 levels 3 weeks of treatment. (b) Plasma IL-7 levels were significantly increased after >3 weeks of IFNβ treatment. A trend toward increased IL-7 levels was seen 18 hours post-injection. *p < 0.05. IFNβ: interferon beta; IL-7Rα: interleukin-7 receptor alpha chain; MS: multiple sclerosis; RQ: relative quantification.
IFNβ-treated MS patients’ T-cells have decreased IL-7Rα expression CD3+ T-cells from eight patients starting on IFNβ-1a treatment were sorted by flow cytometry. Cells were collected before treatment start, 18 hours after first injection and when the treatment should have reached a therapeutic effect, after at least three weeks of treatment (Figure 4(a)). Total mRNA was converted to cDNA, and IL-7Rα expression quantified by semiquantitative real-time PCR. After the initial treatment phase CD3+ T-cells expressed significantly lower of IL-7Rα than before treatment initiation (p < 0.05; Figure 4(a)). No significant difference in expression of the alternatively spliced, soluble isoform of IL-7Rα was seen (data not shown). This down-regulation correlated with increased plasma IL-7 levels (p < 0.05; Figure 4(b)), replicating the observed reduction in IL-7 consumption from our cell culture experiments (Figure 3). Leukocyte and lymphocyte counts are reduced as a result of IFNβ treatment Serum IL-7 concentration has been previously shown to be inversely correlated to peripheral blood leukocyte counts.21 Total leukocyte counts were indeed lower in patients on IFNβ treatment not having NAbs (mean: 5.2 × 106 cells) compared with untreated (mean:
Discussion IFNβ was first administered to MS patients more than 30 years ago because of its antiviral properties.22 Although we still do not know the exact mechanism by which IFNβ elicits its therapeutic effect, the general understanding leans toward regulation of autoreactive immune events. This notion is supported by reduced major histocompatibility complex (MHC) II expression on antigen-presenting cells,23 antiinflammatory skewing of the cytokine balance 12,24 and strengthened integrity of the blood-brain barrier25 in IFNβ-treated patients. It is not, however, entirely surprising that the IFNβ effect on MS pathogenesis is not completely understood, considering that the etiology of MS itself is still unknown. Furthermore, IFNβ’s mechanism of regulating immunity is complex as illustrated by the fact that the first therapeutic injection leads to altered expression of more than 1000 genes.26 Greater understanding of the therapeutic effect of IFNβ may provide clues about the immune-mediated events during MS disease. One locus that is solidly confirmed as an MS susceptibility marker in genetic association studies contains the single nucleotide polymorphism (SNP) rs6897932 located in exon 6 of the IL-7Rα-chain encoding IL7R gene.5,9–11 The biological mechanism behind this (and indeed all other genetic associations in MS) remains debated, but the expression of an alternatively spliced, soluble isoform of the receptor chain is clearly affected by IL7R genotype.9,27,28 We recently showed that increased soluble IL-7Rα levels can potentiate the bioactivity of IL-7 and thereby provide a more pro-inflammatory cytokine milieu.29 This mechanism may affect autoimmunity in general, since rs6897932, or other IL7R SNPs in high linkage disequilibrium, have been associated with a number of autoimmune conditions including Type 1 diabetes and ulcerative
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Peripheral blood leukocyte, lymphocyte and monocyte counts were determined in MS patients. (a) IFNβ-treated patients who had not developed anti-drug antibodies (IFNβ (NAb–); n = 25) had significantly lower leukocyte counts per ml blood (mean 5.2 × 106) compared with untreated (mean 7.5 × 106; n = 16) and natalizumab-treated patients (mean 7.8 × 106; n = 18). (b) A similar pattern was seen in lymphocyte counts. IFNβ (NAb–) patients had significantly lower mean lymphocytes per ml blood (1.6 × 106) than untreated (mean 2 × 106) and natalizumab-treated patients (mean 3.5 × 106). There was also a significant increase in lymphocyte counts among natalizumab-treated patients compared with untreated patients. (c) No significant difference on circulating monocyte numbers between IFNβ (NAb–) (mean 0.41 × 106), IFNβ (NAb+) or untreated MS patients was seen; however, natalizumab-treated patients had higher monocyte counts (mean 0.67 × 106). *p < 0.05. MS: multiple sclerosis; IFNβ: interferon beta; NAb: neutralizing antibodies.
Figure 5. MS patients’ blood counts are influenced by treatment.
W Lundström, C Hermanrud et al.
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Multiple Sclerosis Journal 20(13) colitis.17 Given IL-7’s central role in T-cell development and survival and its involvement in autoimmune processes,30,31 it is plausible that IL-7 aside from being critical for a well-functioning immune system can promote autoreactivity in certain settings. In this work we demonstrate a strong biological link between the most widely used first-line treatment of MS (IFNβ) and the only cytokine-receptor pair genetically associated with disease susceptibility (IL7 and IL7R). The elevated serum IL-7 levels seen as a result of IFNβ administration (Figure 1) were mitigated by NAb (Figure 2), underlining the direct link between IFNβ bioactivity and serum IL-7 concentration. Moreover, a switch in treatment from IFNβ to natalizumab induced an average ~three-fold decrease in systemic IL-7 on an individual level, showing that the effect depends on treatment rather than disease. We further show that IFNβ directly affects IL-7Rα expression coupled with reduced IL-7 consumption in vitro (Figure 3(c)), and elevated serum IL-7 concentration in vivo (Figure 4(b)). We also saw that IFNβ treatment led to reduced leukocyte and lymphocyte values from complete blood counts (Figure 5), in accordance with previous findings.32,33 To what extent this is caused by impaired IL-7 responsiveness remains to be determined. It is known, however, that IL-7 is non-redundant for both T-cell development and survival, and hence plays a key role in maintaining the peripheral lymphocyte pool. Our results suggest that this axis is utilized, at least partially, by IFNβ to induce mild leukopenia. Reduced lymphocyte counts may also contribute to further elevated serum IL-7 concentration. Previous reports have seen an inverse correlation between lymphocyte counts and IL-7 concentration in response to human immunodeficiency virus (HIV),34 idiopathic CD4+ T-cell lymphocytopenia35 and high-dose chemotherapy in cancer.36 While this study clearly shows that IFNβ modulates IL-7 levels by affecting IL-7Rα expression, a secondary effect of IFNβ on the reduction of white blood cells may lead to a further increase in IL-7. With our increasing understanding of IL-7’s importance in autoimmunity in general, and MS in particular, IL-7 signaling has become a topic that warrants further scrutiny. Our work demonstrates a more than three-fold increase in systemic IL-7 among patients undergoing IFNβ treatment compared with natalizumab. It needs to be investigated thoroughly whether chronically elevated levels of IL-7 can diminish the efficacy of IFNβ by providing a pro-inflammatory milieu in patients already experiencing autoimmune
disease. Furthermore, IL-7 has been recently shown to up-regulate the target molecule of natalizumab (CD49d), which is an essential tool used by autoreactive T-cell clones to cross the blood-brain barrier.37 Hypothetically, depleting (or reducing) IL-7 bioactivity in combination with IFNβ administration may in fact augment treatment efficacy and would be relevant to explore further. Furthermore, future investigations of the findings presented in this manuscript should include genetic correlations with IL7 and IL7R genotype given their impact on disease susceptibility. Acknowledgements The authors thank Ingegerd Löfving Arvholm and Anna Mattsson for technical assistance with NAb testing, and Ramus Gustafsson for critical review of this manuscript. Conflicts of interest Tomas Olsson has received advisory board honoraria and lecture fees, as well as unrestricted MS research grants, from Genzyme, Biogen Idec, Novartis and Merck. Jan Hillert has received honoraria for serving on advisory boards for Biogen Idec, Merck Serono and Novartis, and for speaker’s fees from BiogenIdec, Merck-Serono, Bayer-Schering, Teva and SanofiAventis. He has served as P.I. for and received projects supported by Biogen Idec, Merck-Serono and Bayer-Schering. Virginija Karrenbauer has received a speaker’s fee from Merck-Serono and a scholarship from Biogen-Idec. The other authors have nothing to declare. Funding Jan Hillert’s MS research is funded by the Swedish Research Council, Bibbi and Nils Jensens Foundation and the European Commission. Anna Fogdell-Hahn and Christina Hermanrud have received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n° 115303, resources of which are composed of financial bution from the European Union’s Seventh contri Framework Program (FP7/2007–2013) and EFPIA companies’ in-kind contribution.
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16. Cohen BA, Oger J, Gagnon A, et al. The implications of immunogenicity for protein-based multiple sclerosis therapies. J Neurol Sci 2008; 275: 7–17. 17. Lundström W, Fewkes NM and Mackall CL. IL-7 in human health and disease. Semin Immunol 2012; 24: 218–224. 18. Bertolotto A, Sala A, Caldano M, et al. Development and validation of a real time PCR-based bioassay for quantification of neutralizing antibodies against human interferon-beta. J Immunol Methods 2007; 321: 19–31. 19. Jungedal R, Lundkvist M, Engdahl E, et al. Prevalence of anti-drug antibodies against interferon beta has decreased since routine analysis of neutralizing antibodies became clinical practice. Mult Scler 2012; 18: 1775–1781. 20. Kawade Y. Quantitation of neutralization of interferon by antibody. Methods Enzymol 1986; 119: 558–573. 21. Fry TJ, Connick E, Falloon J, et al. A potential role for interleukin-7 in T-cell homeostasis. Blood 2001; 97: 2983–2990. 22. Jacobs L, O’Malley J, Freeman A, et al. Intrathecal interferon reduces exacerbations of multiple sclerosis. Science 1981; 214: 1026–1028. 23. Arnason BG. Interferon beta in multiple sclerosis. Clin Immunol Immunopathol 1996; 81: 1–11. 24. Ozenci V, Kouwenhoven M, Huang YM, et al. Multiple sclerosis is associated with an imbalance between tumour necrosis factor-alpha (TNF-alpha)and IL-10-secreting blood cells that is corrected by interferon-beta (IFN-beta) treatment. Clin Exp Immunol 2000; 120: 147–153. 25. Stone LA, Frank JA, Albert PS, et al. The effect of interferon-beta on blood-brain barrier disruptions demonstrated by contrast-enhanced magnetic resonance imaging in relapsing–remitting multiple sclerosis. Ann Neurol 1995; 37: 611–619. 26. Sellebjerg F, Datta P, Larsen J, et al. Gene expression analysis of interferon-beta treatment in multiple sclerosis. Mult Scler 2008; 14: 615–621. 27. Crawley AM, Faucher S and Angel JB. Soluble IL-7R alpha (sCD127) inhibits IL-7 activity and is increased in HIV infection. J Immunol 2010; 184: 4679–4687. 28. Hoe E, McKay FC, Schibeci SD, et al. Functionally significant differences in expression of diseaseassociated IL-7 receptor alpha haplotypes in CD4 T cells and dendritic cells. J Immunol 2010; 184: 2512–2517. 29. Lundström W, Highfill S, Walsh ST, et al. Soluble IL7Rα potentiates IL-7 bioactivity and promotes autoimmunity. Proc Natl Acad Sci U S A 2013; 110: E1761–E1770.
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research-article2014
MSJ0010.1177/1352458514532700Multiple Sclerosis JournalW Lundström, C Hermanrud
MULTIPLE SCLEROSIS MSJ JOURNAL
Research Paper
Interferon beta treatment of multiple sclerosis increases serum interleukin-7 Wangko Lundström, Christina Hermanrud, Maria Sjöstrand, Susanna Brauner, Marie WahrenHerlenius, Tomas Olsson, Virginija Karrenbauer, Jan Hillert and Anna Fogdell-Hahn
Multiple Sclerosis Journal 2014, Vol. 20(13) 1727–1736 DOI: 10.1177/ 1352458514532700 © The Author(s), 2014. Reprints and permissions: http://www.sagepub.co.uk/ journalsPermissions.nav
Abstract Background: Interleukin-7 (IL-7) is a non-redundant cytokine for T-cell development and survival. The IL-7 signaling pathway has been genetically and functionally associated with several autoimmune diseases including multiple sclerosis (MS). Objective: The objective of this paper is to elucidate the effect of the widely used immunomodulatory MS therapy interferon beta (IFNβ) on IL-7 homeostasis. Methods: Swedish MS patients were screened for IL-7 concentration in serum and blood cell counts. IL-7 receptor alpha chain (IL-7Rα) expression was determined by semi-quantitative real-time polymerase chain reaction (PCR) and flow cytometry. Results: IFNβ treatment led to significantly increased serum IL-7 levels (mean: 17 pg/ml) compared with healthy controls (mean: 7.6 pg/ml) and natalizumab-treated patients (mean: 5.3 pg/ml). In vitro and in vivo, peripheral blood leukocytes showed decreased IL-7Rα expression and IL-7 consumption upon IFNβ exposure, suggesting that their IL-7 responsiveness is impaired during treatment. Conclusions: MS patients undergoing IFNβ treatment have increased serum IL-7 levels and decreased IL-7 consumption. Given IL-7’s important role in T-cell immunity, this relationship may be highly relevant for IFNβ’s treatment efficacy.
Keywords: Multiple sclerosis, immunology, interferon beta, natalizumab Date received: 16 September 2013; revised: 10 March 2014; accepted: 30 March 2014
Introduction The exact cause of multiple sclerosis (MS) remains unknown, but thanks to twin- and migration studies it is clear that both genetic and environmental factors contribute to determine disease risk.1 Human leukocyte antigen (HLA) was the first genetic region associated with MS susceptibility, and HLA-DRB1*15 remains the strongest known genetic predisposition.2–5 Over the last few years the list of MS susceptibility linked genes has grown rapidly with the introduction of genomewide association studies (GWAS). Although explaining a substantial part of MS causality, the genotypes that increase susceptibility seem to have no impact on disease severity, suggesting separate mechanisms regulating disease onset versus progress.6–8 The first confirmed genetic association to MS susceptibility outside the HLA region was in the interleukin-7 receptor gene (IL7R).9–11
IL7R encodes the IL-7 receptor alpha chain (IL-7Rα or CD127), which forms the IL-7 receptor when in complex with the common gamma chain (CD132), and the thymic stromal lymphopoietin receptor (TSLPR) when in complex with the TSLPR chain. Apart from the IL7R association, a recent multicenter GWAS confirmed the IL7 gene as a genetic modulator of MS susceptibility.5 Interferon beta (IFNβ) was the first approved drug for treatment of relapsing–remitting MS and has been used as a first-line disease-modifying agent over the past two decades. The exact mechanism by which IFNβ modulates MS progress remains unknown, but immune modulation is widely accepted as, at least part of, the explanation.12,13 Treatment with recombinant human IFNβ has showed both decreased relapse rate and reduced size
Correspondence to: Anna Fogdell-Hahn Center for Molecular Medicine, Karolinska Institutet, SE-17176 Stockholm, Sweden. [email protected] Wangko Lundström Christina Hermanrud Tomas Olsson Virginija Karrenbauer Jan Hillert Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Maria Sjöstrand Susanna Brauner Marie Wahren-Herlenius Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Sweden
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Multiple Sclerosis Journal 20(13) of brain lesions,14 hence early treatment start could potentially reduce disability.15 Three types of IFNβ preparations are currently available for treatment of MS: intramuscular (IM) IFNβ-1a (Avonex, Biogen Idec, Weston, MA, USA), subcutaneous (SC) IFNβ-1a (Rebif 22/44 µg, Serono, Geneva, Switzerland) and SC IFNβ-1b (Betaferon/Extavia, Bayer/Novartis, Basel, Switzerland). Up to 47 percent of patients being treated with IFNβ will develop neutralizing antibodies (NAbs), which can abrogate the bioactivity of the drug.16 Presence of NAbs is therefore an important factor to consider when determining proper therapy for MS patients. Over the past few years, IL-7 and its receptor have been shown to influence autoimmunity in various diseases and animal models.17 In order to optimize MS treatment with IFNβ, it is important to understand its impact on endogenous immune processes. In this work we present in vitro and in vivo investigations elucidating the interplay between IFNβ administration and IL-7 biology. Materials and methods Study group Serum samples from 184 Swedish MS patients receiving IFNβ treatment and tested for NAbs at the NAb routine laboratory at Karolinska Institutet were included in this study. Patients were treated with different IFNβ preparations, either IFNβ-1a (Avonex, n = 73; Rebif 22 μg, n = 10; Rebif 44 μg, n = 58), or IFNβ-1b (Betaferon, n = 43). Serum samples were collected one to seven days post-IFNβ injection. Patients with or without NAbs to IFNβ were included in the study. The control cohorts were composed of serum samples retrieved from six healthy volunteers and 21 natalizumab-treated MS patients. Serum samples from healthy volunteers were obtained from the Karolinska Hospital blood bank and MS patients on natalizumab therapy from the NAb routine laboratory at Karolinska Institutet, and all were NAb negative. All patients on natalizumab therapy had previously been treated with IFNβ and been on natalizumab treatment for at least six months. For IL7R gene expression analysis, serial blood samples were collected from eight MS patients, who were treatment naïve before study entry. Samples were taken from each patient before IM IFNβ-1a treatment onset, approximately 18 hours after the first injection, and three to 13 weeks post-treatment start-up and 12–18 hours after last injection.
Data from the Immunomodulation and MS Epidemiology (IMSE) cohort at Karolinska University Hospital, Huddinge was retrieved for MS patients treated with IFNβ or natalizumab. Leukocyte, lymphocyte and monocyte populations as well as whole blood counts were quantified by flow cytometry at a clinical laboratory using the XE-5000 system (Sysmex co, Bremerhaven, DE). The values of leukocyte, lymphocyte, and monocyte numbers were grouped as follows: before IFNβ treatment start (n = 16), during IFNβ treatment (NAb– n = 25; NAb+ n = 12), and during natalizumab treatment (n = 18). For a summary of patient demographics, see Supplementary Table 1. NAb analysis MS patients were screened for the presence of antiIFNβ antibody neutralizing activity using the myxovirus resistance protein A (MxA) gene expression bioassay (MGA).18,19 Patient serum was incubated with A549 cells, and the ability of patient sera to neutralize the added recombinant IFNβ-1a (Avonex®, Biogen Idec Inc, Cambridge, MA, USA) was quantified by 7500 Real-Time polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA, USA). Antibody neutralizing activity titers were adjusted according to the Kawade formula20 using Softmax Pro software (Sunnyvale, CA, USA). The NAb titers were expressed as tenfold reduction units per milliliter (TRU/ml) and the patients were classified according to the following categories: negative (1280 TRU/ml). Cell isolation from buffy coat and cell cultures Peripheral blood mononuclear cells (PBMC) from whole blood buffy coats from healthy blood donors (Karolinska Hospital blood bank) were isolated by Ficoll-Hypaque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. PBMCs were counted using a hemocytometer and resuspended to a concentration of 1 × 107 cells/ml and cryopreserved until used. The viability of the cells was determined by trypan blue. A total of 1 × 106 cells/ml of cryopreserved PBMCs were cultured in medium consisting of Roswell Park Memorial Institute (RPMI) 1640 medium (GIBCO, Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal calf serum (Thermo Fisher, Waltham, MA, USA), and 1% penicillinstreptomycin (Life Technologies) at 37°C in 5% CO2. Flow cytometry PBMCs were cultured at 106 cells/ml in 400 µl medium per well of a 24-well plate (NUNC) with
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W Lundström, C Hermanrud et al. recombinant IFNβ-1a (Avonex®, Biogen Idec Inc, Cambridge, MA, USA) at various concentrations (1000, 100, 10, 1, 0.1, 0 IU/ml). After one, three, five, and seven days of incubation, cells were stained with CD8-PB (Life Technologies), CD3APC, CD4-PerCP, CD14-fluorescein isothiocyanate (FITC) (BD Biosciences, Franklin Lakes, NJ, USA), CD19-PE and CD127-PE-Cy7 (BioLegend, San Diego, CA, USA) at 4°C for 30 minutes. The samples were acquired on a CyAn Dako flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed by FlowJo software (Tree Star, Ashland, OR, USA) and GraphPad Prism (La Jolla, CA, USA). Data are representative of three independent experiments. IL-7 receptor-mediated IL-7 consumption PBMCs from healthy volunteers were cultured in 200 µl medium per well in a 96-well plate (NUNC) with recombinant human IL-7 (Roskilde, Denmark) at various concentrations (1000, 100, 10, 0 pg/ml) with or without recombinant IFNβ-1a (Avonex®, Biogen Idec Inc) at 1000 IU/ml. After four, 21, 45, 72 and 216 hours of incubation, cells were counted and IL-7 levels in the culture medium were quantified by a commercially available enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (HS IL-7 ELISA kit, R&D Systems, Minneapolis, MN, USA). Data are representative of three independent experiments. Real-time quantitative PCR Total RNA from CD3+ fluorescence-activated cell sorted (FACS) T-cells was isolated using Trizol (Life Technologies) according to the manufacturer’s protocol. To enhance RNA precipitation 50 µg of glycogen (Thermo Fisher) was added. The RNA concentration was determined by NanoDrop (Thermo Fisher). RNA was reverse transcribed to cDNA by iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Expression level of full length membrane-bound IL-7Rα (presence of exon 6) and the soluble form of IL-7Rα (absence of exon 6) was measured using C1000 Thermal Cycler (Bio-Rad) commercially available primer-probe solutions (Life Technologies; full-length IL-7Rα: Hs00904814_m1, sIL-7Rα: Hs00902337_m1, and HPRT1: Hs01003267_m1). Expression levels were normalized to HPRT1. Expression values were calculated using the 2ΔΔCt formula where Ct values were the mean of technical duplicates. Statistical analyses Statistical calculations were performed using Prism software (GraphPad Inc). p values were calculated by
unpaired, two-sided t tests and statistical significance was defined as a p value < 0.05. Ethical considerations Informed consent for all parts of the study were obtained from all patients and studies were approved by the regional ethics board in Stockholm. Results IFNβ treatment leads to elevated serum IL-7 levels in MS patients To assess the potential impact of IFNβ treatment on IL-7 homeostasis, we first determined serum IL-7 levels in 205 MS patients and six healthy blood donors by ELISA. Patients receiving IFNβ treatment (n = 184) had significantly increased IL-7 levels 3 weeks of treatment. (b) Plasma IL-7 levels were significantly increased after >3 weeks of IFNβ treatment. A trend toward increased IL-7 levels was seen 18 hours post-injection. *p < 0.05. IFNβ: interferon beta; IL-7Rα: interleukin-7 receptor alpha chain; MS: multiple sclerosis; RQ: relative quantification.
IFNβ-treated MS patients’ T-cells have decreased IL-7Rα expression CD3+ T-cells from eight patients starting on IFNβ-1a treatment were sorted by flow cytometry. Cells were collected before treatment start, 18 hours after first injection and when the treatment should have reached a therapeutic effect, after at least three weeks of treatment (Figure 4(a)). Total mRNA was converted to cDNA, and IL-7Rα expression quantified by semiquantitative real-time PCR. After the initial treatment phase CD3+ T-cells expressed significantly lower of IL-7Rα than before treatment initiation (p < 0.05; Figure 4(a)). No significant difference in expression of the alternatively spliced, soluble isoform of IL-7Rα was seen (data not shown). This down-regulation correlated with increased plasma IL-7 levels (p < 0.05; Figure 4(b)), replicating the observed reduction in IL-7 consumption from our cell culture experiments (Figure 3). Leukocyte and lymphocyte counts are reduced as a result of IFNβ treatment Serum IL-7 concentration has been previously shown to be inversely correlated to peripheral blood leukocyte counts.21 Total leukocyte counts were indeed lower in patients on IFNβ treatment not having NAbs (mean: 5.2 × 106 cells) compared with untreated (mean:
Discussion IFNβ was first administered to MS patients more than 30 years ago because of its antiviral properties.22 Although we still do not know the exact mechanism by which IFNβ elicits its therapeutic effect, the general understanding leans toward regulation of autoreactive immune events. This notion is supported by reduced major histocompatibility complex (MHC) II expression on antigen-presenting cells,23 antiinflammatory skewing of the cytokine balance 12,24 and strengthened integrity of the blood-brain barrier25 in IFNβ-treated patients. It is not, however, entirely surprising that the IFNβ effect on MS pathogenesis is not completely understood, considering that the etiology of MS itself is still unknown. Furthermore, IFNβ’s mechanism of regulating immunity is complex as illustrated by the fact that the first therapeutic injection leads to altered expression of more than 1000 genes.26 Greater understanding of the therapeutic effect of IFNβ may provide clues about the immune-mediated events during MS disease. One locus that is solidly confirmed as an MS susceptibility marker in genetic association studies contains the single nucleotide polymorphism (SNP) rs6897932 located in exon 6 of the IL-7Rα-chain encoding IL7R gene.5,9–11 The biological mechanism behind this (and indeed all other genetic associations in MS) remains debated, but the expression of an alternatively spliced, soluble isoform of the receptor chain is clearly affected by IL7R genotype.9,27,28 We recently showed that increased soluble IL-7Rα levels can potentiate the bioactivity of IL-7 and thereby provide a more pro-inflammatory cytokine milieu.29 This mechanism may affect autoimmunity in general, since rs6897932, or other IL7R SNPs in high linkage disequilibrium, have been associated with a number of autoimmune conditions including Type 1 diabetes and ulcerative
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Peripheral blood leukocyte, lymphocyte and monocyte counts were determined in MS patients. (a) IFNβ-treated patients who had not developed anti-drug antibodies (IFNβ (NAb–); n = 25) had significantly lower leukocyte counts per ml blood (mean 5.2 × 106) compared with untreated (mean 7.5 × 106; n = 16) and natalizumab-treated patients (mean 7.8 × 106; n = 18). (b) A similar pattern was seen in lymphocyte counts. IFNβ (NAb–) patients had significantly lower mean lymphocytes per ml blood (1.6 × 106) than untreated (mean 2 × 106) and natalizumab-treated patients (mean 3.5 × 106). There was also a significant increase in lymphocyte counts among natalizumab-treated patients compared with untreated patients. (c) No significant difference on circulating monocyte numbers between IFNβ (NAb–) (mean 0.41 × 106), IFNβ (NAb+) or untreated MS patients was seen; however, natalizumab-treated patients had higher monocyte counts (mean 0.67 × 106). *p < 0.05. MS: multiple sclerosis; IFNβ: interferon beta; NAb: neutralizing antibodies.
Figure 5. MS patients’ blood counts are influenced by treatment.
W Lundström, C Hermanrud et al.
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Multiple Sclerosis Journal 20(13) colitis.17 Given IL-7’s central role in T-cell development and survival and its involvement in autoimmune processes,30,31 it is plausible that IL-7 aside from being critical for a well-functioning immune system can promote autoreactivity in certain settings. In this work we demonstrate a strong biological link between the most widely used first-line treatment of MS (IFNβ) and the only cytokine-receptor pair genetically associated with disease susceptibility (IL7 and IL7R). The elevated serum IL-7 levels seen as a result of IFNβ administration (Figure 1) were mitigated by NAb (Figure 2), underlining the direct link between IFNβ bioactivity and serum IL-7 concentration. Moreover, a switch in treatment from IFNβ to natalizumab induced an average ~three-fold decrease in systemic IL-7 on an individual level, showing that the effect depends on treatment rather than disease. We further show that IFNβ directly affects IL-7Rα expression coupled with reduced IL-7 consumption in vitro (Figure 3(c)), and elevated serum IL-7 concentration in vivo (Figure 4(b)). We also saw that IFNβ treatment led to reduced leukocyte and lymphocyte values from complete blood counts (Figure 5), in accordance with previous findings.32,33 To what extent this is caused by impaired IL-7 responsiveness remains to be determined. It is known, however, that IL-7 is non-redundant for both T-cell development and survival, and hence plays a key role in maintaining the peripheral lymphocyte pool. Our results suggest that this axis is utilized, at least partially, by IFNβ to induce mild leukopenia. Reduced lymphocyte counts may also contribute to further elevated serum IL-7 concentration. Previous reports have seen an inverse correlation between lymphocyte counts and IL-7 concentration in response to human immunodeficiency virus (HIV),34 idiopathic CD4+ T-cell lymphocytopenia35 and high-dose chemotherapy in cancer.36 While this study clearly shows that IFNβ modulates IL-7 levels by affecting IL-7Rα expression, a secondary effect of IFNβ on the reduction of white blood cells may lead to a further increase in IL-7. With our increasing understanding of IL-7’s importance in autoimmunity in general, and MS in particular, IL-7 signaling has become a topic that warrants further scrutiny. Our work demonstrates a more than three-fold increase in systemic IL-7 among patients undergoing IFNβ treatment compared with natalizumab. It needs to be investigated thoroughly whether chronically elevated levels of IL-7 can diminish the efficacy of IFNβ by providing a pro-inflammatory milieu in patients already experiencing autoimmune
disease. Furthermore, IL-7 has been recently shown to up-regulate the target molecule of natalizumab (CD49d), which is an essential tool used by autoreactive T-cell clones to cross the blood-brain barrier.37 Hypothetically, depleting (or reducing) IL-7 bioactivity in combination with IFNβ administration may in fact augment treatment efficacy and would be relevant to explore further. Furthermore, future investigations of the findings presented in this manuscript should include genetic correlations with IL7 and IL7R genotype given their impact on disease susceptibility. Acknowledgements The authors thank Ingegerd Löfving Arvholm and Anna Mattsson for technical assistance with NAb testing, and Ramus Gustafsson for critical review of this manuscript. Conflicts of interest Tomas Olsson has received advisory board honoraria and lecture fees, as well as unrestricted MS research grants, from Genzyme, Biogen Idec, Novartis and Merck. Jan Hillert has received honoraria for serving on advisory boards for Biogen Idec, Merck Serono and Novartis, and for speaker’s fees from BiogenIdec, Merck-Serono, Bayer-Schering, Teva and SanofiAventis. He has served as P.I. for and received projects supported by Biogen Idec, Merck-Serono and Bayer-Schering. Virginija Karrenbauer has received a speaker’s fee from Merck-Serono and a scholarship from Biogen-Idec. The other authors have nothing to declare. Funding Jan Hillert’s MS research is funded by the Swedish Research Council, Bibbi and Nils Jensens Foundation and the European Commission. Anna Fogdell-Hahn and Christina Hermanrud have received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n° 115303, resources of which are composed of financial bution from the European Union’s Seventh contri Framework Program (FP7/2007–2013) and EFPIA companies’ in-kind contribution.
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