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Leukemia inhibitory factor tips the immune balance towards regulatory T cells in multiple sclerosis

Brain, Behavior, and Immunity, pages 180 - 188

Highlights

 

  • LIF treatment may provide neuroprotection and remyelination to treat early and progressive MS.
  • The LIF receptor is upregulated on immune cells of MS patients.
  • LIF boosts the number of Tregsin vitroin donors with low serum levels of IL-6.
  • LIF treatment enhances Treg numbers and ameliorates symptoms in a preclinical MS model.

Abstract

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), for which current treatments are unable to prevent disease progression. Based on its neuroprotective and neuroregenerating properties, leukemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) cytokine family, is proposed as a novel candidate for MS therapy. However, its effect on the autoimmune response remains unclear. In this study, we determined how LIF modulates T cell responses that play a crucial role in the pathogenesis of MS. We demonstrate that expression of the LIF receptor was strongly increased on immune cells of MS patients. LIF treatment potently boosted the number of regulatory T cells (Tregs) in CD4+T cells isolated from healthy controls and MS patients with low serum levels of IL-6. Moreover, IL-6 signaling was reduced in the donors that responded to LIF treatmentin vitro. Our data together with previous findings revealing that IL-6 inhibits Treg development, suggest an opposing function of LIF and IL-6. In a preclinical animal model of MS we shifted the LIF/IL-6 balance in favor of LIF by CNS-targeted overexpression. This increased the number of Tregs in the CNS during active autoimmune responses and reduced disease symptoms. In conclusion, our data show that LIF downregulates the autoimmune response by enhancing Treg numbers, providing further impetus for the use of LIF as a novel treatment for MS and other autoimmune diseases.

Keywords: Multiple sclerosis, Therapeutics, Leukemia inhibitory factor, Regulatory T cells.

1. Introduction

Multiple sclerosis (MS) is a chronic disabling disease of the central nervous system (CNS), characterized by multifocal inflammatory infiltrates, demyelination, axonal loss and neurodegeneration. Autoreactive T helper 1 (Th1) and Th17 cells are thought to be the main effector cells in MS, whereas regulatory T cells (Tregs) function to maintain self-tolerance and prevent autoimmunity ( Broux et al., 2013 ). Loss of suppressive function of circulating CD4+Tregs is demonstrated in MS patients (Haas et al, 2005, Venken et al, 2008, and Viglietta et al, 2004). As a result of the regulatory defects, Th1 and Th17 cells are hyperactive and cross the blood–brain barrier to initiate an inflammatory response leading to demyelinated lesions. Alternatively, Th cells could be further educated regionally to become either regulatory or pathogenic depending on the route of entry to the CNS ( Baruch and Schwartz, 2013 ). Available therapies suppress the immune response, however they mainly moderate the initial relapsing-remitting phase and are unable stop disease progression ( Lopez-Diego and Weiner, 2008 ). Therefore, there is a high need for novel therapies that modulate the immune response and provide neuroprotection or even promote neural regeneration. Leukemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) cytokine family, has been proposed as a promising candidate for MS therapy (Metcalfe, 2011a, Metcalfe, 2011b, and Slaets et al, 2010a), as it promotes survival of neurons and oligodendrocytes, and stimulates neurite outgrowth (Leibinger et al, 2009, Slaets et al, 2008, and Vanderlocht et al, 2006). In an animal model of MS, experimental autoimmune encephalomyelitis (EAE), LIF treatment ameliorates clinical symptoms by preventing demyelination (Butzkueven et al, 2002 and Slaets et al, 2010b) and by limiting axonal damage and loss ( Gresle et al., 2012 ). Furthermore, in a toxin-induced demyelination model LIF stimulates proliferation of oligodendrocyte progenitor cells and enhances hippocampal remyelination ( Deverman and Patterson, 2012 ).

Besides these promising neuroprotective and repair promoting effects in preclinical models of MS, LIF may also directly modulate the autoimmune response thereby providing dual disease ameliorating mechanisms. LIF induces an anti-inflammatory phenotype in macrophages (Duluc et al, 2007 and Hendriks et al, 2008). Moreover, in studies on graft rejection LIF was associated with transplantation tolerance, while its family member IL-6 was associated with allo-rejection ( Gao et al., 2009 ). In contrast to LIF, IL-6 is a potent pro-inflammatory cytokine driving Th17 differentiation (Acosta-Rodriguez et al, 2007, Bettelli et al, 2006, and Volpe et al, 2008).

The immunomodulatory properties of LIF remain largely unknown and are a hurdle for its application in the clinic. In this study, the therapeutic potential of LIF is further revealed by studying its effect on the T cell compartment. We reveal that in MS patients the LIF receptor (LIFR) was strongly increased on circulating T cells. Moreover, while LIF did not promote Th1, Th2 and Th17 differentiationin vitro, it enhanced the number of Tregs in donors with low serum levels of IL-6. Therapeutic application in EAE doubled the number of Tregs in the CNS and ameliorated clinical symptoms. Our combined human andin vivomouse data reveal that LIF downregulates the autoimmune response and provide novel treatment strategies for MS patients.

2. Materials and methods

2.1. Study subjects

For measuring LIFR expression, blood samples were collected from 22 healthy controls, 41 untreated and 43 treated patients with clinically definite MS ( Table 1 ). Patients received treatment with interferon β (IFNβ; Avonex®, Rebif®, Betaferon®), glatiramer acetate (Copaxone®) or Natalizumab (Tysabri®). Healthy controls, untreated and treated MS patients were age and gender matched ( Table 1 ). For additionalin vitroassays, blood samples of 15 healthy controls and 12 untreated MS patients were used. Blood samples were collected in collaboration with the University Biobank Limburg (UBiLim). This study was approved by the Medical Ethical Committee of the University Hospital K.U. Leuven and informed consent was obtained from all study subjects. For immunohistochemistry, frozen brain tissue from 4 chronic active MS patients was obtained from the Netherlands Brain Bank (NBB, Amsterdam, Netherlands).

Table 1 Study subjects used for analysis of the LIFR on circulating immune cells.

  Treated MS patients Untreated MS patients Healthy controls
Number n = 43 n = 41 n = 22
Age (years) 45.09 46.63 39.59
Male/female ratio 15/28 (0.54) 13/28 (0.46) 9/13 (0.69)
Disease duration (years) 12.00 12.37 NA
EDSS 3.35 3.87 NA
 
MS type
 RR 31 23 NA
 CP 9 15 NA
 
Treatment
 IFNβ 24 NA NA
 Glatiramer acetate 8 NA NA
 Natalizumab 11 NA NA

MS, multiple sclerosis; EDSS, expanded disability status scale; RR, relapsing remitting; CP, chronic progressive; IFNβ; interferon beta; NA, not applicable.

2.2. EAE induction

Female 10 week old C56BL/6J mice (Harlan, Horst, the Netherlands) were immunized subcutaneously with myelin oligodendrocyte glycoprotein 35–55 peptide (MOG3555) emulsified in complete Freund’s adjuvant (CFA) containing Mycobacterium tuberculosis according to manufacturer’s guidelines (Hooke Laboratories, Lawrence, USA). Directly after immunization and 24 h later, mice were intraperitoneally injected with pertussis toxin. Mice were daily weighted and evaluated for neurological signs of disease using a standard 5-point scale; 0: no symptoms; 1: limp tail; 2: hind limp weakness; 3: complete hind limp paralysis; 4: complete hind limp paralysis and partial front leg paralysis; 5: death. All animal procedures were in accordance with the EU Directive 2010/63/EU for animal experiments and were approved by the Hasselt university ethics committee.

2.3. Lentiviral vector injection

Lentiviral vector encoding LIF (LV-LIF) and LV encoding enhanced green fluorescent protein (eGFP) were constructed as described in Slaets et al. (2010b) . When mice showed EAE symptoms for 3 to 4 days they were stereotactically injected with LV-LIF or LV-eGFP in the right lateral ventricle. Mice were anesthetized with ketamine (75 mg/kg) and medetomidine (1 mg/kg), after which they were placed in a stereotactic head frame (Stoelting, IL, USA), a midline incision of the skin was made and a small hole was drilled in the skull. At a rate of 0.25 μl/min 4 μl of concentrated vector (4.8 * 107 pg p24/ml) was injected using a 10 μl Hamilton syringe with 30-gauge needle. Coordinates for injection into the lateral ventricle were anteroposterior 0.02 cm, lateral 0.1 cm and dorsoventral −0.18 cm using bregma as reference.

2.4. Cell culture and T helper cell differentiation

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using density gradient centrifugation (Cedarlane lympholyte, Sheffield, UK). PBMCs were cultured in RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS; Hyclone Europe, Erembodegem, Belgium), 1% nonessential amino acids, 1% sodium pyruvate, 50 U/ml penicillin and 50 mg/ml streptomycin (all Life technologies, Merelbeke, Belgium). To measure LIFR expression after activation and IFNβ treatment, cells were stimulated with 2 μg/ml anti-CD3 (clone 2G3, BIOMED, Diepenbeek, Belgium), 2 μg/ml CpG2006 (ODN2006, InvivoGen, Toulouse, France) and 1000 U/ml recombinant human IFNβ (R&D systems, Abingdon, UK).

For differentiation toward Th1, Th2 and Th17 cells, CD4+T cells were isolated from PBMCs by immunomagnetic isolation (Easysep, Stemcell technologies, Grenoble, France). CD4+T cells were activated with anti-CD3/CD28 beads (Life technologies). Cytokines and neutralizing antibodies to induce differentiation were added: for Th1 differentiation 10 ng/ml IL-12 and 5 μg/ml anti-IL-4 antibody, for Th2 differentiation 200 ng/ml IL-4, 5 μg/ml anti-IFNγ antibody and 5 μg/ml anti-IL-12 antibody (Affymetrix, Vienna, Austria) and for Th17 differentiation 25 ng/ml IL-23, 5 μg/ml anti-IL-4 and 5 μg/ml anti-IFNγ antibody (all R&D systems). 25 ng/ml LIF (Millipore, Overijse, Belgium) was added alone or in combination with the differentiation-inducing cytokines and neutralizing antibodies.

To determine the effect of LIF on Tregs, memory CD4+T cells (CD45RO+) were isolated from PBMCs using magnetic bead labeling (Miltenyi Biotec, Leiden, The Netherlands). Cells were treated with 25 or 250 ng/ml LIF (Millipore) for 3 days.

2.5. Flow cytometry

To determine LIFR expression, isolated PBMCs were incubated with anti-gp130-FITC (Abcam, Cambridge, UK) and anti-LIFRβ-PE antibodies (R&D systems), combined with PerCP labeled antibodies specific for the immune cell subsets, CD3, CD4, CD8, CD14 and CD19 (BD Biosciences, Erembodegem, Belgium). For FoxP3 staining memory CD4+T cells were surface stained with anti-CD25-FITC (BD Biosciences), followed by fixation and permeabilization using human Foxp3 buffer set (BD Bioscience) and intracellular staining using anti-Foxp3-PE (BD Biosciences). Individuals were classified as responders when a minimal increase in the percentage of FOXP3+CD25highTregs of 1.2 was measured. Samples were run on FACSCalibur and analyzed using CellQuest Software (BD Biosciences). To determine the extent of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) after IL-6 treatment,isolated memory CD4+T cells were allowed to rest for 2 h, followed by incubation with 25 ng/ml IL-6 (BD Biosciences) for 15 min. Cells were fixated and permeabilized using Cytofix™ fixation buffer and Phosflow™ Perm Buffer III, respectively, and intracellular staining was performed with anti-pSTAT3-Alexa Fluor 647 (BD Biosciences). Fluorescence intensity was measured using FACSAriaII and analyzed using FACSDiva software 6.1.3 (BD Biosciences).

Mononuclear cells were isolated from the spinal cord of EAE mice 7 days after stereotactic injection of LV-LIF or LV-eGFP. Briefly, animals were transcardially perfused with Ringer’s solution. Spinal cords were dissected and incubated in RPMI containing 175 U/ml collagenase (Sigma–Aldrich, Bornem, Belgium) for 1 h at 37 °C, followed by 30% percoll density centrifugation (GE Healthcare). Single cell suspensions were obtained by passing the samples through a 70 μm filter. To determine the number of Tregs, cells were surface stained using anti-CD4-PerCP (BD biosciences) and anti-CD25-Alexa Fluor 488 antibodies (Affymetrix), followed by fixation and permeabilization using Mouse Foxp3 buffer set (BD Bioscience) and intracellular staining with anti-Foxp3-PE (BD Biosciences). To measure IFNγ, IL-17 and IL-4 cells were stimulated for 4 h with 25 ng/ml phorbol-12-myristate-13-acetate (PMA), 1 μg/ml calcium ionomycin (both Sigma–Aldrich) and Golgiplug (BD Biosciences). Surface staining was performed with anti-CD4-PerCP (BD Biosciences), followed by fixation and permeabilization using the Cytofix/Cytoperm™ Solution Kit (BD Biosciences) and intracellular staining with anti-IFNγ-APC, IL-17-PE and IL-4-Alexa Fluor 488 (all Affymetrix). FACSAriaII was used to measure fluorescence intensity and FACSDiva software 6.1.3 for analysis (BD Biosciences).

2.6. Immunohistochemistry

Ten micrometer cryosections of 4 MS patients were cut on the Leica CM3050S cryostat (Leica Microsystems, Wetzlar, Germany). For 3,3′ diaminobenzidine (DAB) staining, cryosections were fixed in acetone, blocked with 10% goat serum and incubated with human LIFRβ antibody (Santa Cruz biotechnology, Dallas, Texas). Binding of the antibody was visualized using a goat anti-rabbit HRP-labeled antibody, followed by DAB substrate (both Dako, Heverlee, Belgium). For fluorescent staining sections were fixated, blocked and incubated with antibodies against human LIFRβ, IBA-1 (Wako, Neuss, Germany) and CD4 (AbD serotec, Düsseldorf, Germany). Binding of these primary antibodies was visualized with the appropriate Alexa 488 or 555-conjugated secondary antibody (Life technologies) and nuclear staining was performed with DAPI (Life technologies). Autofluorescence was blocked using 0,3% Sudan Black in 70% ethanol.

Mice were transcardially perfused with Ringer’s solution 20 days after LV injection, spinal cords were dissected and snap frozen in liquid nitrogen. Ten micrometer sections were made using Leica CM3050S cryostat. Sections were fixed in acetone, blocked with Protein Block Dakocytomation (Dako) and incubated with anti-mouse CD3 and F4/80 antibodies (AbD serotec). Immunoreactivity was visualized using Alexa 555 secondary antibodies and nuclear staining was performed with DAPI (both Life technologies). Microscopical analysis was performed using an Eclipse 80i microscope (Nikon, Amstelveen, the Netherlands) and for image collection the Nis-Elements Basic Research version 2.3 microscopy software was used.

2.7. Real-time quantitative PCR

RNA was prepared from the Th1, Th2 and Th17 cultures using the High Pure RNA Isolation Kit (Roche, Almere, The Netherlands) according to manufacturer’s instructions. Conversion of RNA to cDNA was performed using the Reverse Transcription System (Promega, Leiden, The Netherlands). Quantitative PCR was conducted on a StepOnePlus Real-Time PCR detection system (Applied Biosystems, Gaasbeek, Belgium) using universal cycling conditions (20 s at 95 °C, 40 cycles of 3 s at 95 °C and 30 s at 60 °C). The PCR reaction consisted of fast SYBR green master mix (Applied Biosystems), 10 μM of forward and reverse primers (Eurogentec, Seraing, Belgium), RNase free water and 12.5 ng template cDNA. The expression was normalized using the two most stable reference genes (CyCA and GAPDH) and converted to fold change as compared to cells only incubated in culture medium using comparative Ct method. Primers used for quantitative PCR are shown in Table SI .

2.8. ELISA

Concentration of IL-6 in the plasma of healthy controls and MS patients was measured using a commercially available quantitative human IL-6 ELISA (eBiosciences) according to manufacturer’s instructions. Plasma was collected from the blood samples used to measure percentages of Tregs after LIF treatment.

2.9. Statistical analysis

Statistical analysis of LIFR expression of healthy controls, treated and untreated MS patients was performed using a non-parametric Mann–Whitney test. Repeated measures ANOVA was used to determine LIFR expression after different time points of activation. Dunn’s multiple comparison test was used to define the effect of LIF treatment on the percentage of FOXP3+CD25highT cells. Unpairedt-test was used to reveal differences in LIFR expression, pSTAT3 and IL-6 serum concentration between responders and non-responder MS patients, and differences in the number of CD3, F4/80, Foxp3, IFNγ, IL-17 and IL-4-positive cells between LV-eGFP and LV-LIF treated mice. Differences in EAE scores were analyzed using Wilcoxon matched pairs test. Analyses were performed using GraphPad Prism version 5.01.

3. Results

3.1. In MS patients LIFR expression is strongly upregulated on T cells, B cells and monocytes

To define whether LIF is able to modulate the ongoing autoimmune response in MS patients, we analyzed which circulating immune cell subsets express the LIFR and compared this to healthy controls. LIF binds to the LIFRβ subunit, which then recruits gp130 to form a heterodimer that induces signaling. Expression of LIFRβ and gp130 was measured by flow cytometry on immune cells isolated from the blood. In MS patients, higher percentages of CD3+T cells (19.92% vs 1.91%), CD4+T helper cells (19.06% vs 2.29%), CD8+cytotoxic T cells (16.51% vs 1.86%), CD19+B cells (11.18% vs 5.02%) and CD14+monocytes (62.82% vs 37.30%) express LIFRβ-gp130 as compared to healthy controls ( Fig. 1 A–E). When analyzing both subunits separately, gp130 is consistently expressed on all immune cells in healthy controls, while the LIFRβ subunit is only expressed by low percentages of immune cells ( Fig. S1A–E ). In MS patients however, the LIFRβ subunit is strongly upregulated on the different immune cells subsets ( Fig. S1A–E ). These data show that gp130 is constitutively expressed and that in MS patients expression of the specific LIFRβ subunit is augmented on circulating immune cells.

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Fig. 1 Expression of the LIFR is highly increased on immune subsets of MS patients. (A–E) Percentage of CD3+T cells (A), CD4+T helper cells (B), CD8+cytotoxic T cells (C), CD19+B cells (D) and CD14+monocytes (E) co-expressing LIFRβ and gp130 were analyzed in healthy controls (n = 22) and untreated MS patients (n = 41) using flow cytometry. (F–H) LIFRβ-gp130 expression was measured on CD4+T helper cells (F), CD8+cytotoxic T cells (G) and CD19+B cells (H) of healthy subjects (n = 4) after activation of PBMCs with anti-CD3 antibody (2 μg/ml) (F and G) or CpG (2 μg/ml) (H). (N) Percentage CD4+T cells expressing LIFRβ-gp130 following activation of PBMCs of healthy donors for 3 days (T72) with anti-CD3 antibody in absence or presence of IFN-β (1000 U/ml) (n = 8). Percentages are compared to expression levels at day 0 (T0). Data are depicted as mean ± SEM. (I–M) Percentage of CD3+(I), CD4+(J), CD8+(K), CD19+(L) and CD14+cells (M) expressing LIFRβ-gp130 in treated (n = 43) and untreated (n = 41) MS patients measured using flow cytometry. Dots represent the percentage of positive cells in each donor and bars represent median per group.p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001. LIFRβ, leukemia inhibitory factor receptor beta; gp130, glycoprotein 130; MS, multiple sclerosis; HC, healthy controls; α-CD3, anti-CD3 antibody.

To determine whether activation of immune cells is responsible for the upregulated LIFR expression, we stimulated PBMCs with anti-CD3 antibody to activate T cells or with CpG to activate B cells. Activation of T or B cells induced a clear increase in LIFRβ-gp130 expression ( Fig. 1 F–H). Next, we defined whether current MS therapies, such as IFNβ, glatiramer acetate and natalizumab, aimed to suppress aberrant immune responses also downregulate LIFR expression. All immunosuppressive treatments strongly reduced the percentage of circulating immune cells expressing LIFRβ-gp130 as compared to untreated MS patients: CD3+T cells (1.59% vs 19.92%), CD4+T helper cells (1.29% vs 19.06%), CD8+cytotoxic T cells (1.93% vs 16.51%), CD19+B cells (3.63% vs 11.18%) and CD14+monocytes (27.95% vs 62.82%) ( Fig. 1 I–M). The reduction in receptor expression was detected for both subunits, LIFRβ and gp130 ( Fig. S1F–J ) and no difference in receptor expression was found between the different treatments ( Fig. S1K–O ). Taken together, these data suggest that activation of the immune system in MS patients enhances LIFR expression, while immunosuppressive treatment lowers receptor expression. To confirm this, T cells were ex vivo activated in absence or presence of the immunosuppressive agent IFNβ. Indeed, activation of T cells induced significant upregulation of LIFRβ-gp130, while activation of IFNβ-treated T cells did not lead to a significant increase ( Fig. 1 N).

To determine whether augmented LIFR expression is also evident on infiltrated autoreactive immune cells in MS lesions, we analyzed LIFRβ expression in post-mortem MS lesion material using immunohistochemistry. In the lesion, and especially in the active rim of the lesion, strong expression of LIFRβ was found ( Fig. 2 A). Macrophages and microglia ( Fig. 2 B) were positive for the receptor; as confirmed by a double staining with IBA-1 ( Fig. 2 C). In addition, we found a small subset of perivascular CD4+T helper cells expressing LIFRβ ( Fig. 2 D). Thus, during autoimmune disease circulating and CNS infiltrating immune subsets strongly upregulate their LIFR making these cells susceptible to LIF signaling.

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Fig. 2 In MS lesions, LIFRβ is expressed by macrophages/microglia and perivascular T cells. Immunohistochemical analysis for the LIFRβ was performed on brain sections of chronic active MS patients (n = 4). (A, magnification B) Strong LIFRβ expression was found in the lesion and especially in the active rim. (C and D) Fluorescent double staining for LIFRβ (green) and IBA-1 (C) or CD4 (D) (red) was also performed on these brain sections. Magnification of the double positive cells is shown in the right corner. LIFRβ, leukemia inhibitory factor receptor beta; NAWM, normal appearing white matter. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. LIF enhances Treg numbers in human memory CD4+ T cells depending on the LIF/IL-6 balance

As CD4+T cells are considered to play an essential role in the pathogenesis of MS, we unraveled the effect of LIF on the differentiation of the different T helper subtypes. CD4+T cells were isolated from the blood of healthy subjects and differentiated toward Th1, Th2 and Th17 cells. Co-incubation with LIF did not affect differentiation toward Th1, Th2 and Th17 cells, neither did LIF treatment itself induce Th1, Th2 or Th17 cells ( Fig. 3 A–F). In contrast, treatment of memory CD4+T cells with LIF for 3 days increased the number of FOXP3+CD25highTregs in a subpopulation of healthy controls and untreated MS patients ( Fig. 4 A and B). Similar responses were detected in healthy donors and MS patients, 40% of the healthy controls responded to LIF (4/10) and 33% of the MS patients (4/12). As FOXP3 upregulation can also be observed upon activation of T cells ( Wang et al., 2007 ), we measured demethylation of CpG nucleotides located in the first intron of FOXP3 (FOXP3i1), which is a highly specific marker for human Tregs ( Lucas et al., 2012 ). A positive correlation between the percentage of FOXP3+CD25highTregs and the percentage of cells with demethylated FOXP3i1 was observed in LIF-treated CD4+T cells ( Fig. S2 ).

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Fig. 3 LIF does not affect differentiation of human CD4+T cells toward Th1, Th2 and Th17 cells. (A–F) CD4+T cells were isolated from the blood of healthy subjects (n = 5), after which they were incubated with IL-12 (10 ng/ml) and anti-IL-4 antibody (5 μg/ml) to induce Th1 differentiation (A and B), IL-4 (200 ng/ml), anti-IFNγ (5 μg/ml) and anti-IL-12 antibody (5 μg/ml) for Th2 differentiation (C and D), and IL-23 (25 ng/ml), anti-IL-4 (5 μg/ml) and anti-IFNγ antibody (5 μg/ml) for Th17 differentiation (E and F). LIF (25 ng/ml) was added to these cultures with and without the differentiation-inducing cytokines and neutralizing antibodies. mRNA was isolated from these cells to measure Th1 differentiation factors, IFNγ (A) and Tbet (B); Th2 factors, IL-4 (C) and GATA3 (D), and Th17 factors, IL-17 (E) and RORγt (F) using QPCR. The expression was normalized using the two most stable reference genes and converted to fold change as compared to cells only incubated in culture medium using the 2−ΔΔCTmethod. LIF, leukemia inhibitory factor; IL, interleukin; IFNγ, interferon gamma.

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Fig. 4 LIF increases the number of Tregs in memory CD4+T cells of healthy controls and untreated MS patients with low IL-6 serum levels.(A and B) Memory CD4+T cells were isolated from the blood of healthy subjects (n = 10) and untreated MS patients (n = 12) and were incubated with 25 or 250 ng/ml LIF for 3 days. The number of FOXP3+CD25highcells was measured using flow cytometry. Subjects were classified as responders when a minimal increase in the number of FOXP3+CD25highcells of 1.2 was detected. Of the 10 healthy controls, 4 were classified as responders and 6 as non-responders. In the untreated MS patients, 4 were responders and 8 non-responders. Data are expressed as mean per group as compared to control (0 ng/ml LIF). (C) LIFR-gp130+cells were measured in CD4+T cells and CD4+CD25highCD127lowTregs of the responding and non-responding donors by flow cytometry. (D and E) Memory CD4+T cells of responders and non-responders were treated with 25 ng/ml IL-6 for 15 min and the percentage of cells positive for pSTAT3 was measured using flow cytometry. (F) The concentration of IL-6 was measured in plasma of responders and non-responders by ELISA. Data are expressed as mean ± SEM,p < 0.05. LIFRβ, leukemia inhibitory factor receptor beta; gp130, glycoprotein 130; pSTAT3, phosphorylated signal transducer and activator of transcription 3.

Next, we aimed to reveal why some individuals responded to LIF and others did not by determining differences in LIFR expression and signaling in responders vs non-responders. Responders displayed a trend towards higher percentages of CD4+T helper cells expressing LIFRβ-gp130 as compared to non-responders (p-value = 0.0505, Fig. 4 C). In addition, significantly more responder-derived CD4+CD25highCD127lowTregs expressed LIFRβ-gp130 ( Fig. 4 C). Previous studies demonstrated that its family member IL-6 inhibits Treg developmentin vitroas well asin vivo(Bettelli et al, 2006, Fujimoto et al, 2011, Korn et al, 2008, and Mangan et al, 2006). Moreover, it is suggested that LIF and IL-6 oppose each other in inducing Th17 differentiation ( Metcalfe, 2011b ). To reveal whether there is an influence of IL-6 on LIF responsiveness, we assessed IL-6 signaling in the responders and non-responders via the Janus kinases/signal transducer and activator of transcription (JAK/STAT) pathway by measuring pSTAT3. IL-6 treatment of memory CD4+T cells of the LIF responders resulted in a reduced percentages of cells positive for pSTAT3 compared to the non-responders ( Fig. 4 D and E). Additionally, in the non-responders higher serum levels of IL-6 were detected, while in the responders low IL-6 levels were measured ( Fig. 4 F). Taken together, whilein vitroLIF treatment does not affect Th1, Th2 or Th17 differentiation, it enhances Tregs in subjects with low serum levels of IL-6.

3.3. Therapeutic LIF administration boosts the number of CNS infiltrated Tregs and ameliorates EAE symptoms

Our human data suggest that shifting the LIF/IL-6 balance in favor of LIF increases the number of Tregs and could thus restore immune tolerance and consequently limit the inflammatory damage. We tested this hypothesis in EAE, a preclinical mouse model of MS, by therapeutic overexpression of LIF using LVs. LVs were intrathecally injected thereby transducing especially the ependymal cells lining the cerebrospinal fluid-filled spaces and choroid plexus cells ( Slaets et al., 2010b ), resulting in a continuous secretion of LIF in the cerebrospinal fluid and spread throughout the CNS. EAE was induced in C57BL/6J mice by immunization with MOG3555. Three days after clinical onset, when IL-6 levels are highly upregulated ( Fig. S3 ), mice were stereotactically injected with LV-LIF or LV-eGFP. CNS-targeted LIF expression after disease onset significantly reduced EAE symptoms as compared to LV-eGFP injected mice ( Fig. 5 A). In the control group disease symptoms increased, while in the LIF-treated mice there was no increase and the mice recovered more rapidly. No difference in the number of CD3+infiltrating T cells or F4/80+macrophages/microglia was found in the spinal cord after the second relapse ( Fig. 5 B–E). Interestingly, CNS-targeted LIF treatment doubled the percentage of FOXP3+CD25+Tregs ( Fig. 5 F and J) and reduced the percentage of IFNγ-producing Th1 cells present in the spinal cord 7 days after LV injection ( Fig. 5 G and K). No effect of LIF treatment was detected on IL4+Th2 and IL17+Th17 cells ( Fig. 5 H, I and K). These findings demonstrate that favoring the balance to LIF during autoimmune disease increases the number of Tregs in the CNS and reduces disease burden.

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Fig. 5 CNS-targeted LIF expression after disease onset induces a regulatory phenotype in infiltrating T cells and ameliorates EAE symptoms. C57BL/6J mice were immunized with MOG3555in CFA. When mice displayed EAE symptoms for 3–4 days LV-LIF or LV-eGFP was stereotactically injected into the right lateral ventricle. (A) Clinical scores in the LV-LIF (n = 7) and LV-eGFP-injected mice (n = 6), representative of 3 independent experiments. (B–E) Quantification and representative pictures of the number of infiltrating CD3+T cells (B and C) and F4/80+macrophages/microglia (D and E) measured using immunohistochemistry in the spinal cord at day 20 after vector injection in LIF (n = 7) and eGFP mice (n = 6). (F–K) Quantification and representative dot plots of the number of Tregs (CD4+CD25+FOXP3+, F and J), Th1 cells (CD4+IFNγ+, G and K), Th17 cells (CD4+IL17+, H and K) and Th2 cells (CD4+IL4+, I) in the spinal cord at day 7 after injection with LV-LIF (n = 8) or LV-eGFP (n = 8) using flow cytometry. The percentage of cells was measured as percentage of positive cells within the CD4+gate. Data are expressed as mean ± SEM,p < 0.05 and∗∗∗p < 0.001. EAE, experimental autoimmune encephalomyelitis; LV-LIF, lentiviral vector encoding leukemia inhibitory factor; LV-eGFP, LV encoding enhanced green fluorescent protein; IL, interleukin; IFNγ, interferon gamma.

4. Discussion

LIF treatment is a promising therapeutic strategy to protect neurons and oligodendrocytes from inflammatory damage in MS lesions and to promote remyelination, a strategy that would enable treatment of progressive MS patients. Here, we show that this cytokine can also limit autoimmune-mediated damage by enhancing Treg numbers.

First, we demonstrated a highly upregulated LIFR expression on circulating T cells, B cells and monocytes of untreated MS patients. The gp130 subunit was ubiquitously expressed on all immune cell subsets, while the specific LIFRβ subunit was expressed on low percentages of immune cells in healthy controls but strongly enhanced in MS patients. Moreover, we revealed that activation of immune cells upregulates the LIFR, while current immunosuppressive treatments reduce receptor expression. In active lesions of MS patients, LIFRβ was expressed on macrophages/microglia and perivascular T cells. Elevated levels of LIFRβ are also reported on neurons in the motor cortex of MS patients ( Dutta et al., 2007 ). Moreover, LIF itself is increased in the serum, cerebrospinal fluid and lesions of MS patients (Mashayekhi and Salehi, 2011 and Vanderlocht et al, 2006). We believe that the upregulation of the LIFR and its ligand in MS patients is an endogenous response to heal inflammatory CNS lesions based on our findings that LIF downregulates aberrant immune responses by increasing Tregs numbers, in addition to its established neuroprotective properties.

We demonstrated that LIF enhanced the number of Tregs in healthy controls and MS patients who had reduced serum levels of IL-6. Moreover, in the LIF responders reduced IL-6 induced pSTAT3 signaling was found. Studies on graft rejection in mice already demonstrated that LIF is associated with transplantation tolerance and supports the expression of FOXP3 (Gao et al, 2009 and Park et al, 2011). IL-6, in contrast, limits the generation of Tregsin vitroandin vivoin mice (Bettelli et al, 2006 and Korn et al, 2008).Both family members signal through gp130, IL-6 binds the IL-6Rα subunit which recruits a homodimer of gp130, whereas the LIFR is composed of a LIFRβ and gp130 subunit ( Heinrich et al., 2003 ). IL-6 was previously shown to strongly inhibit transcription of the LIFRβ subunit ( Gao et al., 2009 ), which further supports their opposing function and may explain the reduced LIFR expression in the CD4+T cell compartment of the non-responders.

We further reveal that CNS directed overexpression of LIFin vivodoubled Treg numbers and reduced clinical symptoms of EAE. In parallel, the number of Th1 cells was reduced, whereas no effect on Th17 cell number was detected. Whether the LIF induced expansion of Tregs directly suppressed local Th1 cells is difficult to proof in our setup. If indeed Tregs would directly suppress pathogenic Th cells in the spinal cord, a similar reduction of Th17 cells would be expected, which was not the case. Alternatively, infiltrating Th1 cells may be re-educated locally and adapt to a regulatory phenotype under influence of locally provided cues, including LIF. Both the cytokine signature encountered by infiltrating T cells and the route of entry to the CNS may orchestrate CNS immunity. Indeed, Schwartz and colleagues recently showed that complex interactions of CNS-specific T effector and regulatory cells with the choroid plexus are able to instruct resolution of neuroinflammation ( Raposo et al., 2014 ). Together, our data suggest that LIF can restore immune tolerance leading to reduced inflammatory damage and allowing CNS repair.

This study revealed that LIF is a promising therapeutic candidate as besides the previously reported neuroprotective and neuroregenerating properties it can also suppress the autoimmune response. Some concerns regarding the application of LIF in the clinic still have to be resolved. Phase I studies demonstrated the short half-life of LIF in serum indicating that repetitive injections would be needed (Goggin et al, 2004 and Gunawardana et al, 2003).In addition, the limited potential to cross the blood–brain barrier means that high doses need to be administered to reach the target organ, risking systemic side effects. Delivery approaches such as LIF-loaded nanoparticles directed against CD4+T cells can increase efficiency of LIF treatment as shown in a non-human primate model ( Park et al., 2011 ). Alternatively, CNS-targeted LIF expression by means of lentiviral vectors would maximize neuroprotection, in addition to modulating the infiltrating immune cells. Based on our findings, the potency of LIF as a therapy may be further augmented by combining it with compounds that block IL-6 signaling.

In conclusion, we are the first to show that LIF enhances the number of Tregsin vitroin humans. Moreover, LIF treatment in a preclinical model of MS augments Treg numbers during active autoimmune responses and ameliorated clinical symptoms. The beneficial effects of LIF on the immune system demonstrated in this study in combination with the neuroprotective and neuroregenerating properties reported earlier provide great opportunities for the treatment of MS patients in the early as well as the progressive stage of the disease.

Acknowledgments

This work was financially supported by grants from the Flemish Fund for Scientific Research (FWO Vlaanderen, G04441N), the Interuniversity Attraction Poles (IUAP-P7-39), the Belgian MS-Liga, Methusalem NEURONET and BOF-UHasselt.

Appendix A. Supplementary data

 

fx1

Supplementary Fig. S1 The LIFRβ subunit is strongly upregulated on immune subsets of untreated MS patients, while treatment reduces receptor expression. (A–E) Expression of the subunits LIFRβ and gp130 separately on CD3+T cells (A), CD4+T helper cells (B), CD8+cytotoxic T cells (C), CD19+B cells (D) and CD14+monocytes (E) in healthy controls (n = 22) and untreated MS patients (n = 41). (F–J) Expression of the subunits, LIFRβ and gp130, on CD3+(F), CD4+(G), CD8+(H), CD19+(I) and CD14+cells (J) in untreated (n = 41) and treated MS patients (n = 43). (K–O) Percentage of CD3+(K), CD4+(L), CD8+(M), CD19+(N) and CD14+cells (O) expressing LIFR-gp130 were analyzed in healthy controls (n = 22), untreated (n = 41), IFNβ (n = 24), glatiramer acetate (n = 8) and natalizumab (n = 11) treated MS patients using flow cytometry. Dots represent the percentage of positive cells in each donor and bars represent median per group.p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001, LIFRβ, LIF receptor beta; gp130, glycoprotein 130; MS, multiple sclerosis; HC, healthy control; GA, glatiramer acetate.

fx2

Supplementary Fig. S2 The percentage of FOXP3+CD25highTregs positively correlates to the percentage of FOXP3i1 demethylation. FOXP3i1 demethylation was measured in CD4+memory T cells treated with 25 ng/ml LIF for 3 days (n = 8) and correlated to the percentages of FOXP3+CD25highTregs (p-value = 0.0279,r2 = 0.5031). FOXP3i1, CpG nucleotides located in the first intron of FOXP3.

fx3

Supplementary Fig. S3 IL-6 is highly augmented during EAE, with a peak in expression two days after disease onset. C57BL/6J mice were immunized with MOG35-55in CFA. At onset (day 2), at peak of disease symptoms (day 5) and in the chronic phase (day 16), spinal cords were isolated (n = 5 per group). mRNA expression of IL-6 was measured by QPCR, normalized using the two most stable reference genes and converted to fold change as compared to spinal cords of healthy mice using the 2−ΔΔCTmethod. Data are expressed as mean ± SEM,∗∗∗p < 0.001. EAE, experimental autoimmune encephalomyelitis.

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Supplementary material

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Supplementary Table S1 Primers for quantitative PCR of human Th1, Th2 and Th17 cultures.

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Footnotes

a Department of Immunology, Biomedical Research Institute, Hasselt University, Agoralaan Building C, 3590 Diepenbeek, Belgium

b Laboratory for Neurobiology and Gene Therapy, Katholieke Universiteit Leuven, Kapucijnenvoer 33, 3000 Leuven, Belgium

c de Duve Institute, Université Catholique de Louvain, Avenue Hippocrate 75, 1200 Brussels, Belgium

d Department of Molecular Cell Biology and Immunology, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

e Revalidatie & MS-Centrum, Boemerangstraat 2, 3900 Overpelt, Belgium

lowast Corresponding author. Tel.: +32 11 26 92 68.


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