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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
- 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.
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.
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).
|Treated MS patients||Untreated MS patients||Healthy controls|
|Number||n = 43||n = 41||n = 22|
|Male/female ratio||15/28 (0.54)||13/28 (0.46)||9/13 (0.69)|
|Disease duration (years)||12.00||12.37||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 (MOG35–55) 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).
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 .
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.
3.1. 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.
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.
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 ).
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 MOG35–55. 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.
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.
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.
- Acosta-Rodriguez et al., 2007 E.V. Acosta-Rodriguez, G. Napolitani, A. Lanzavecchia, F. Sallusto. Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Nat. Immunol.. 2007;8:942-949 Crossref
- Baruch and Schwartz, 2013 K. Baruch, M. Schwartz. CNS-specific T cells shape brain function via the choroid plexus. Brain Behav. Immun.. 2013;34:11-16 Crossref
- Bettelli et al., 2006 E. Bettelli, Y. Carrier, W. Gao, T. Korn, T.B. Strom, M. Oukka, H.L. Weiner, V.K. Kuchroo. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235-238 Crossref
- Broux et al., 2013 B. Broux, P. Stinissen, N. Hellings. Which immune cells matter? The immunopathogenesis of multiple sclerosis. Crit. Rev. Immunol.. 2013;33:283-306 Crossref
- Butzkueven et al., 2002 H. Butzkueven, J.G. Zhang, M. Soilu-Hanninen, H. Hochrein, F. Chionh, K.A. Shipham, B. Emery, A.M. Turnley, S. Petratos, M. Ernst, P.F. Bartlett, T.J. Kilpatrick. LIF receptor signaling limits immune-mediated demyelination by enhancing oligodendrocyte survival. Nat. Med.. 2002;8:613-619 Crossref
- Deverman and Patterson, 2012 B.E. Deverman, P.H. Patterson. Exogenous leukemia inhibitory factor stimulates oligodendrocyte progenitor cell proliferation and enhances hippocampal remyelination. J. Neurosci.. 2012;32:2100-2109 Crossref
- Duluc et al., 2007 D. Duluc, Y. Delneste, F. Tan, M.P. Moles, L. Grimaud, J. Lenoir, L. Preisser, I. Anegon, L. Catala, N. Ifrah, P. Descamps, E. Gamelin, H. Gascan, M. Hebbar, P. Jeannin. Tumor-associated leukemia inhibitory factor and IL-6 skew monocyte differentiation into tumor-associated macrophage-like cells. Blood. 2007;110:4319-4330 Crossref
- Dutta et al., 2007 R. Dutta, J. McDonough, A. Chang, L. Swamy, A. Siu, G.J. Kidd, R. Rudick, K. Mirnics, B.D. Trapp. Activation of the ciliary neurotrophic factor (CNTF) signalling pathway in cortical neurons of multiple sclerosis patients. Brain. 2007;130:2566-2576 Crossref
- Fujimoto et al., 2011 M. Fujimoto, M. Nakano, F. Terabe, H. Kawahata, T. Ohkawara, Y. Han, B. Ripley, S. Serada, T. Nishikawa, A. Kimura, S. Nomura, T. Kishimoto, T. Naka. The influence of excessive IL-6 production in vivo on the development and function of Foxp3+ regulatory T cells. J. Immunol.. 2011;186:32-40 Crossref
- Gao et al., 2009 W. Gao, L. Thompson, Q. Zhou, P. Putheti, T.M. Fahmy, T.B. Strom, S.M. Metcalfe. Treg versus Th17 lymphocyte lineages are cross-regulated by LIF versus IL-6. Cell Cycle. 2009;8:1444-1450 Crossref
- Goggin et al., 2004 T. Goggin, Q.T. Nguyen, A. Munafo. Population pharmacokinetic modelling of Emfilermin (recombinant human leukaemia inhibitory factor, r-hLIF) in healthy postmenopausal women and in infertile patients undergoing in vitro fertilization and embryo transfer. Br. J. Clin. Pharmacol.. 2004;57:576-585 Crossref
- Gresle et al., 2012 M.M. Gresle, E. Alexandrou, Q. Wu, G. Egan, V. Jokubaitis, M. Ayers, A. Jonas, W. Doherty, A. Friedhuber, G. Shaw, M. Sendtner, B. Emery, T. Kilpatrick, H. Butzkueven. Leukemia inhibitory factor protects axons in experimental autoimmune encephalomyelitis via an oligodendrocyte-independent mechanism. PLoS One. 2012;7:e47379 Crossref
- Gunawardana et al., 2003 D.H. Gunawardana, R.L. Basser, I.D. Davis, J. Cebon, P. Mitchell, C. Underhill, T.J. Kilpatrick, K. Reardon, M.D. Green, P. Bardy, P. Amor, D. Crump, S. Ng, R.L. Nation, C.G. Begley. A phase I study of recombinant human leukemia inhibitory factor in patients with advanced cancer. Clin. Cancer Res.. 2003;9:2056-2065
- Haas et al., 2005 J. Haas, A. Hug, A. Viehover, B. Fritzsching, C.S. Falk, A. Filser, T. Vetter, L. Milkova, M. Korporal, B. Fritz, B. Storch-Hagenlocher, P.H. Krammer, E. Suri-Payer, B. Wildemann. Reduced suppressive effect of CD4+CD25high regulatory T cells on the T cell immune response against myelin oligodendrocyte glycoprotein in patients with multiple sclerosis. Eur. J. Immunol.. 2005;35:3343-3352 Crossref
- Heinrich et al., 2003 P.C. Heinrich, I. Behrmann, S. Haan, H.M. Hermanns, G. Muller-Newen, F. Schaper. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem. J.. 2003;374:1-20 Crossref
- Hendriks et al., 2008 J.J. Hendriks, H. Slaets, S. Carmans, H.E. de Vries, C.D. Dijkstra, P. Stinissen, N. Hellings. Leukemia inhibitory factor modulates production of inflammatory mediators and myelin phagocytosis by macrophages. J. Neuroimmunol.. 2008;204:52-57 Crossref
- Korn et al., 2008 T. Korn, M. Mitsdoerffer, A.L. Croxford, A. Awasthi, V.A. Dardalhon, G. Galileos, P. Vollmar, G.L. Stritesky, M.H. Kaplan, A. Waisman, V.K. Kuchroo, M. Oukka. IL-6 controls Th17 immunity in vivo by inhibiting the conversion of conventional T cells into Foxp3+ regulatory T cells. Proc. Natl. Acad. Sci. U.S.A.. 2008;105:18460-18465 Crossref
- Leibinger et al., 2009 M. Leibinger, A. Muller, A. Andreadaki, T.G. Hauk, M. Kirsch, D. Fischer. Neuroprotective and axon growth-promoting effects following inflammatory stimulation on mature retinal ganglion cells in mice depend on ciliary neurotrophic factor and leukemia inhibitory factor. J. Neurosci.. 2009;29:14334-14341 Crossref
- Lopez-Diego and Weiner, 2008 R.S. Lopez-Diego, H.L. Weiner. Novel therapeutic strategies for multiple sclerosis – a multifaceted adversary. Nat. Rev. Drug Discovery. 2008;7:909-925
- Lucas et al., 2012 S. Lucas, N. van Baren, C. de Smet, P.G. Coulie. Demethylation of the FOXP3 gene in human melanoma cells precludes the use of this epigenetic mark for quantification of Tregs in unseparated melanoma samples. Int. J. Cancer. 2012;130:1960-1966 Crossref
- Mangan et al., 2006 P.R. Mangan, L.E. Harrington, D.B. O’Quinn, W.S. Helms, D.C. Bullard, C.O. Elson, R.D. Hatton, S.M. Wahl, T.R. Schoeb, C.T. Weaver. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature. 2006;441:231-234 Crossref
- Mashayekhi and Salehi, 2011 F. Mashayekhi, Z. Salehi. Expression of leukemia inhibitory factor in the cerebrospinal fluid of patients with multiple sclerosis. J. Clin. Neurosci.. 2011;18:951-954 Crossref
- Metcalfe, 2011a S.M. Metcalfe. LIF in the regulation of T-cell fate and as a potential therapeutic. Genes Immun.. 2011;12:157-168 Crossref
- Metcalfe, 2011b S.M. Metcalfe. Multiple sclerosis: one protein, two healing properties. Nature. 2011;477:287-288 Crossref
- Park et al., 2011 J. Park, W. Gao, R. Whiston, T.B. Strom, S. Metcalfe, T.M. Fahmy. Modulation of CD4+ T lymphocyte lineage outcomes with targeted, nanoparticle-mediated cytokine delivery. Mol. Pharm.. 2011;8:143-152
- Raposo et al., 2014 C. Raposo, N. Graubardt, M. Cohen, C. Eitan, A. London, T. Berkutzki, M. Schwartz. CNS repair requires both effector and regulatory T cells with distinct temporal and spatial profiles. J. Neurosci.. 2014;34:10141-10155
- Slaets et al., 2008 H. Slaets, D. Dumont, J. Vanderlocht, J.P. Noben, P. Leprince, J. Robben, J. Hendriks, P. Stinissen, N. Hellings. Leukemia inhibitory factor induces an antiapoptotic response in oligodendrocytes through Akt-phosphorylation and up-regulation of 14-3-3. Proteomics. 2008;8:1237-1247 Crossref
- Slaets et al., 2010a H. Slaets, J.J. Hendriks, P. Stinissen, T.J. Kilpatrick, N. Hellings. Therapeutic potential of LIF in multiple sclerosis. Trends Mol. Med.. 2010;16:493-500 Crossref
- Slaets et al., 2010b H. Slaets, J.J. Hendriks, C. Van den Haute, F. Coun, V. Baekelandt, P. Stinissen, N. Hellings. CNS-targeted LIF expression improves therapeutic efficacy and limits autoimmune-mediated demyelination in a model of multiple sclerosis. Mol. Ther.. 2010;18:684-691
- Vanderlocht et al., 2006 J. Vanderlocht, N. Hellings, J.J. Hendriks, F. Vandenabeele, M. Moreels, M. Buntinx, D. Hoekstra, J.P. Antel, P. Stinissen. Leukemia inhibitory factor is produced by myelin-reactive T cells from multiple sclerosis patients and protects against tumor necrosis factor-alpha-induced oligodendrocyte apoptosis. J. Neurosci. Res.. 2006;83:763-774 Crossref
- Venken et al., 2008 K. Venken, N. Hellings, M. Thewissen, V. Somers, K. Hensen, J.L. Rummens, R. Medaer, R. Hupperts, P. Stinissen. Compromised CD4+ CD25(high) regulatory T-cell function in patients with relapsing-remitting multiple sclerosis is correlated with a reduced frequency of FOXP3-positive cells and reduced FOXP3 expression at the single-cell level. Immunology. 2008;123:79-89 Crossref
- Viglietta et al., 2004 V. Viglietta, C. Baecher-Allan, H.L. Weiner, D.A. Hafler. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J. Exp. Med.. 2004;199:971-979 Crossref
- Volpe et al., 2008 E. Volpe, N. Servant, R. Zollinger, S.I. Bogiatzi, P. Hupe, E. Barillot, V. Soumelis. A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses. Nat. Immunol.. 2008;9:650-657 Crossref
- Wang et al., 2007 J. Wang, A. Ioan-Facsinay, E.I. van der Voort, T.W. Huizinga, R.E. Toes. Transient expression of FOXP3 in human activated nonregulatory CD4+ T cells. Eur. J. Immunol.. 2007;37:129-138 Crossref
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
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