Treatment of Experimental Autoimmune Encephalomyelitis by Sustained Delivery of Low-Dose IFN-α

Marcos Vasquez, Marta Consuegra-Fernández, Fernando Aranda, Aitor Jimenez, Shirley Tenesaca, Myriam Fernandez-Sendin, Celia Gomar, Nuria Ardaiz, Claudia Augusta Di Trani, Noelia Casares, Juan Jose Lasarte, Francisco Lozano and Pedro Berraondo
J Immunol August 1, 2019, 203 (3) 696-704; DOI: https://doi.org/10.4049/jimmunol.1801462

Key Points

  • Recombinant adeno-associated virus expresses low and sustained levels of IFN-α.

  • AAV–IFN-α exerts a potent therapeutic effect and low toxicity in an EAE animal model.

Abstract

Multiple sclerosis (MS) is a chronic autoimmune disease with no curative treatment. The immune regulatory properties of type I IFNs have led to the approval of IFN-β for the treatment of relapsing-remitting MS. However, there is still an unmet need to improve the tolerability and efficacy of this therapy. In this work, we evaluated the sustained delivery of IFN-α1, either alone or fused to apolipoprotein A-1 by means of an adeno-associated viral (AAV) system in the mouse model of myelin oligodendrocyte glycoprotein–induced experimental autoimmune encephalomyelitis. These in vivo experiments demonstrated the prophylactic and therapeutic efficacy of the AAV–IFN-α or AAV–IFN-α fused to apolipoprotein A-1 vectors in experimental autoimmune encephalomyelitis, even at low doses devoid of hematological or neurologic toxicity. The sustained delivery of such low-dose IFN-α resulted in immunomodulatory effects, consisting of proinflammatory monocyte and T regulatory cell expansion. Moreover, encephalitogenic T lymphocytes from IFN-α–treated mice re-exposed to the myelin oligodendrocyte glycoprotein peptide in vitro showed a reduced proliferative response and cytokine (IL-17A and IFN-γ) production, in addition to upregulation of immunosuppressive molecules, such as IL-10, IDO, or PD-1. In conclusion, the results of the present work support the potential of sustained delivery of low-dose IFN-α for the treatment of MS and likely other T cell–dependent chronic autoimmune disorders.

Introduction

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease that affects the CNS and is characterized by inflammation, demyelination, and axonal loss. The etiopathogenesis of MS remains incompletely understood as a result of the complex interplay between several genetic, environmental, and infectious factors (1).

Although there is still no effective therapy for MS, IFN-β has been at the forefront of therapeutic options for many years and is widely used as a first-line treatment for the disorder (2). IFN-β belongs to the type I IFN group, which includes IFN-α (with 13 subtypes), IFN-β, IFN-ε, IFN-κ, and IFN-ω in humans. They are pleiotropic cytokines synthesized and released from different cell sources upon danger signal delivery following host–pathogen interactions and display antiviral, antiproliferative, and immunomodulatory activities (3). Based on the latter properties of type I IFNs, IFN-β has been widely used for the treatment of MS (4). Additionally, IFN-α has also shown therapeutic activity in MS patients by reducing brain lesions, as assessed by magnetic resonance imaging, and attacks (4–7). IFN-α and IFN-β bind to the IFNAR1/2 and activate the JAK/STAT signaling pathway. Although both type I IFNs show some differences at the cellular level (8), they display a similar clinical efficacy, and therefore, the rationale behind the treatment of MS patients with IFN-β instead of using IFN-α is a mix of scientific evidence and tradition.

T cells play a critical role in the etiopathogenesis of MS (9). Indeed, autoreactive T cells from the helper 1 (Th1), helper 17 (Th17), γδ, and CD8+ subsets have been implicated in the accompanying inflammation and neurodegeneration. Cytokines released from Th1 and Th17 cells contribute to enhance MHC class II expression on APCs and increase the cytotoxic activity of autoreactive CD8, macrophages, and NK cells. Moreover, Th17 cells seem to be the main driver in the pathogenesis of MS (10).

In contrast, Th2 cytokines are associated with anti-inflammatory effects. In this setting, regulatory T cells (Treg) can inhibit the activity of autoreactive T cells (11). It has been demonstrated that Treg depletion by using anti-CD25 Abs enhanced the severity of symptoms in an experimental autoimmune encephalomyelitis (EAE) model of MS by a mechanism that involves IL-10 (12). In addition, adoptive cell transfer of Tregs conferred significant protection (13).

The mechanisms of action of type I IFNs in MS are complex and involve effects at multiple levels. Type I IFNs may reduce the trafficking of inflammatory cells across the blood–brain barrier, induce apoptosis of autoreactive T cells, increase the levels of anti-inflammatory cytokines (IL-10, IL-4), decrease the levels of proinflammatory cytokines (IL-17, IFN-γ, TNF-α), and modulate the function of Tregs (14).

Several clinical trials have demonstrated that the clinical benefits of IFN-β in MS are dose dependent (15). The highest doses required to achieve clinical efficacy are, however, normally associated with increased side effects and lead to the production of neutralizing Abs (16). Additionally, its therapeutic efficacy depends on frequent/repeated administration to sustain optimal levels of its biological effect (17). Clinical studies performed in healthy volunteers and MS patients have demonstrated that IFN-β given every other day had significantly greater overall biological activity (18–20) and therapeutic effect (21–23) than weekly administration. Thus, the safety and efficacy of IFN-based therapies may be improved with new administration schedules or IFN delivery systems.

To further improve the pharmacokinetic properties of IFN-based therapies, we have developed a strategy based on IFN-α1 expression by adeno-associated viral (AAV) vectors (24). The low immunogenicity, lack of pathogenicity, persistence, and long-term expression of the transgene incorporated into AAV makes this vector a suitable tool for this approach. Moreover, several clinical trials have demonstrated the safety and efficacy of AAV (25). Additionally, we have developed a strategy based on the fusion of IFN-α1 to apolipoprotein A-1 (IFN-α/ApoA1) (26). This fusion protein presents a longer half-life in circulation with no systemic toxicity when high doses of the AAVs are administered for oncotherapeutic purposes (24).

In this study, we evaluated the therapeutic activity of sustained AAV-mediated IFN-α or IFN-α/ApoA1 expression in EAE mice. We demonstrated that long-term exposure to low IFN-α or IFN-α/ApoA1 levels is sufficient to safely prevent and control EAE symptoms in mice by reducing inflammatory infiltrates, tissue damage, and cytokine responses.

Materials and Methods

Animal handling

In vivo experiments were performed in 6–8-wk-old female C57BL/6 mice (∼20 g) purchased from Harlan Laboratories (Barcelona, Spain). The mice were maintained under specific pathogen-free conditions, and the experimental design was approved by the ethics committees for animal testing of University of Barcelona and Universidad de Navarra.

Induction and clinical score of EAE

Anesthetized mice were immunized by s.c. injection of 150 μg of myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 (MOG35–55, MEVGWYRSPFSRVVHLYRNGK; GenScript, Piscataway, NJ), dissolved in 100 μl of PBS and emulsified with 100 μl of CFA (Difco, Detroit, MI) containing 1 mg Mycobacterium tuberculosis H37RA (no. 231131; BD Biosciences). Each mouse received 100 μl of the emulsion per flank. The i.p. doses of 500 ng of Pertussis toxin (Sigma-Aldrich, St Louis, MO) dissolved in PBS were administered at the time of immunization and 48 h later. Animal weight and clinical score were analyzed daily as follows: 0, no clinical signs; 0.5, partial flaccid paralysis of the tail; 1, flaccid paralysis of the tail; 2, weak hind leg paralysis; 3, bilateral hind leg paralysis; 4, hind and foreleg paralysis; 5, moribund; and 6, death.

Recombinant AAVs were administered once retro-orbitally in a total volume of 200 μl. Previously, animals were anesthetized by i.p. injection of a 1:9 (v/v) mixture of xylazine (Rompun 2%, Bayer) and ketamine (Imalgene 500, Merial).

Recombinant mouse IFN-α produced in CHO cells was purchased from Hycult Biotech (Uden, the Netherlands). Ten thousand units were administered via the retroorbital route three times per week.

To analyze the encephalitogenic capacity of spleen cells isolated from AAV–GFP or AAV–IFN-α, mice were immunized and treated with the AAV vectors 4 d later. At day 15, donor mice were sacrificed and splenocytes cultured in complete RPMI at 37°C and 5% CO2 in the presence of 20 μg/ml MOG35–55, 20 ng/ml recombinant mouse IL-12 (PeproTech, London, U.K.), and 10 μg/ml anti-mouse IFN-γ Ab (XMG1.2; Bio X Cell, West Lebanon, NH). Three days later, 10 × 106 cells were inoculated by retroorbital administration to 10-wk-old female C57BL/6 mice. Animal weight and clinical score were analyzed daily.

Production of recombinant AAV vectors

AAV serotype 8 vectors were constructed to express IFN-α, IFN-α/ApoA1, or GFP under the transcriptional control of the elongation factor 1α promoter. AAV vectors were produced by cotransfection of the plasmids pDP8.ape (PlasmidFactory, Bielefeld, Germany) and pAAV encoding IFN-α1, IFN-α/ApoA1 (26), or GFP into HEK-293T cells. For each production, a mix of 20 μg of pAAV, 55 μg of pDP8.ape, and linear PEI MAX 25 kDa (Polysciences, Warrington, PA) was transfected into HEK-293T cells. Forty-eight hour later, AAV were isolated from the cell lysates by ultracentrifugation in OptiPrep Density Gradient Medium (Sigma-Aldrich), and viral DNA was isolated using the High Pure Viral Nucleic Acid Kit (Roche Applied Science, Mannheim, Germany). The titers of viral particles were subsequently determined by real-time quantitative PCR using primers specific to the EF promoter: forward: 5′- GGTGAGTCACCCACACAAAGG-3′ and reverse: 5′- CGTGGAGTCACATGAAGCGA-3′.

Cell isolation

Spleen cell suspensions were obtained in cold RPMI 1640 by mechanically disrupting the tissue through a 70-μm nylon cell strainer (Falcon, Becton Dickinson) with a syringe plunger. RBCs were removed using Ammonium-Chloride-Potassium lysis buffer (Life Technologies, Rockville, MD).

CD4+CD25+ cells were enriched from pooled spleen by anti-mouse FITC-conjugated CD4+ and allophycocyanin-conjugated CD25+ mAbs (eBioscience, San Diego, CA) and purified by FACSAria II cell sorter (BD Biosciences).

ELISA

Serum IFN-α levels were quantified with VeriKine Mouse IFN-α ELISA Kit (PBL Assay Science, Piscataway, NJ) following the manufacturer’s recommendations. The levels of IFN-γ and IL-17A in the supernatant of splenocytes stimulated with MOG35–55 peptide were measured using Mouse IL-17A ELISA Ready-SET-Go! (eBioscience) and BD OptEIA Mouse IFN-γ ELISA Set (BD Biosciences) kits, respectively and according to the manufacturer’s recommendations.

Hemogram

Blood samples were collected on day 52 after immunization in tubes with 0.5% heparin (Mayne Pharma, Mulgrave, Australia) as the final concentration. Hemograms were analyzed using the Drew Scientific HemaVet Hematology Analyzer (CDC Technologies, Oxford, CT) following the manufacturer’s recommendations.

RNA isolation and quantification of mRNA

RNA was isolated from total and sorted CD4+CD25+ splenocytes or spinal cords using the Maxwell 16 Total RNA Purification Kit (Promega, Madison, WI), quantified in a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE), and retrotranscribed (500 ng) to cDNA with Moloney murine leukemia virus reverse transcriptase from Promega, according to the manufacturer’s instructions.

Quantitative real-time PCR was performed with iQ SYBR Green Supermix (Bio-Rad Laboratories) using specific primers for the following: IFN-stimulated gene 15 (Isg15, 5′- GATTGCCCAGAAGATTGGTG-3′ and 5′-TCTGCGTCAGAAAGACCTCA-3′), ubiquitin-specific peptidase 18 (Usp18, 5′- CCAAACCTTGACCATTCACC-3′ and 5′-ATGACCAAAGTCAGCCCATCC-3′), 2′-5′-oligoadenylate synthetase (2′5′Oas, 5′-ACTGTCTGAAGCAGATTGCG-3′ and 5′-TGGAACTGTTGGAAGCAGTC-3′), IL-10 (5′- GGACAACATACTGCTAACCG-3′ and 5′-AATCACTCTTCACCTGCTCC-3′), IDO 1 (Ido, 5′-CCTTGAAGACCACCACATAG-3′ and 5′-AGCACCTTTCGAACATCGTC-3′), programmed cell death protein 1 (5′-ACTGGTCGGAGGATCTTATG-3′ and 5′-ATCTTGTTGAGGTCTCCAGG-3′), CD4 (5′-GAACATCTGTGAAGGCAAAG-3′ and 5′-CAGCAGCAGCAGCAGCAAG-3′), Foxp3 (5′-GTTTCTCAAGCACTGCCAAG-3′ and 5′-TGTGGAAGAACTCTGGGAAG-3′), IFN-γ (5′-TCAAGTGGCATAGATGTGGAA-3′ and 5′-TGGCTCTGCAGGATTTTCATG-3′), IL-17A (5′-CTGTGTCTCTGATGCTGTTG-3′ and 5′-TATCAGGGTCTTCATTGCGG-3′), F4/80 Ag (F4/80, 5′-CCCAGCTTCTGCCACCTGCA-3′ and 5′-GGAGCCATTCAAGACAAAGCC-3′), and ribosomal protein large P0 (RPLP0, 5′-AACATCTCCCCCTTCTCCTT-3′ and 5′-GAAGGCCTTGACCTTTTCAG-3′). As RPLP0 levels remained constant across different experimental conditions, this parameter was used to standardize gene expression. The amount of each transcript was expressed by the Equation 2ΔCt (2ct(RPLP0) − ct(gene)), with ct being the point at which the fluorescence rises significantly above the background levels.

Splenocyte proliferation assays

Splenocytes (1 × 105) were cultured in 96-well plates for 48 h with MOG35–55 (50 μg/ml). During the last 12 h, samples were pulsed with 0.5 μCi of [3H]thymidine and harvested with a Micro β FilterMate-96 Harvester (PerkinElmer). The [3H]thymidine incorporation was measured using an automated TopCount Liquid Scintillation Counter (Packard).

Flow cytometry analyses

Flow cytometry analyses were performed using a FACS Canto II flow cytometer (BD Biosciences). Splenocytes (5 × 105) were stained with anti-mouse allophycocyanin-conjugated CD11b (M1/70; BioLegend), PE-conjugated Ly6G (1A8; BD Biosciences), and FITC-conjugated Ly6C (AL-21; BD Biosciences) for 15 min at 4°C in FACS buffer (PBS plus 5% FBS and 2.5 mM EDTA) and then washed twice and suspended in the same buffer for further flow cytometric analysis. For Treg analyses, splenocytes were stained with FITC-conjugated CD4 (RM4-5; eBioscience), allophycocyanin-conjugated CD25 (PC61.5, eBioscience), and PE-conjugated Foxp3 (FJK-16s; eBioscience) mAbs.

For intracellular IFN-γ and IL-17A cytokine stainings, splenocytes (5 × 105) were stimulated with MOG35–55 (50 μg/ml) for 66 h. During the last 6 h, cells were restimulated with the same amount of MOG35–55 plus GolgiPlug (1 μl/ml; BD Biosciences), stained with PE-conjugated CD4 (RM4-5; BD Biosciences) mAb for 15 min at 4°C, and fixed with Cytofix/Cytoperm solution (BD Biosciences) for 20 min at 4°C. Finally, cells were stained with PE/Cy7-conjugated anti-mouse IL-17A (TC11-18H10; BioLegend) and FITC-conjugated anti-mouse IFN-γ (XMG1.2; BioLegend), respectively, for 30 min at 4°C, washed twice, and suspended in FACS buffer prior to flow cytometry analysis.

Histological analyses

At day 21 postimmunization, mice (n = 4/group) were euthanized, and their spinal cords isolated and fixed in 4% paraformaldehyde for 24 h. The fixed samples were washed with PBS and stored in 70% ethanol at room temperature until processing. Cell infiltration and myelination were analyzed histochemically by H&E and Luxol Fast Blue Staining, respectively. Quantification was performed by selecting white matter in spinal cord sections. Pixel intensity and density were quantified over a threshold color using MATLAB software (magnification 10×).

Tail suspension test

Mice were suspended by the tail with adhesive tape to a horizontal bar so that the head of the mouse was 20 cm above the floor. The time of immobility was measured by direct-eye observation for 6 min. Experiments were conducted by personnel blinded to the treatment conditions. Depression is expressed as the accumulated time of animal immobility in seconds during a 6-min test.

Statistical analyses

Statistical analyses were carried out with Prism software (GraphPad Software). Data are usually presented as ±SD, and means were compared using a Student t test. To compare three groups, one-way ANOVA followed by Bonferroni posttest was used. Both EAE disease scores and weight curves were fitted and analyzed with nonlinear regression to a second-order polynomial (quadratic) model. The survival data were represented in Kaplan–Meier graphs and analyzed using the log-rank test to determine levels of significance. The p values <0.05 were considered significant.

Results

AAVs encoding IFN-α or IFN-α fused to apolipoprotein A-1 prevents EAE symptoms

Recombinant AAV vectors were used in vivo to evaluate whether sustained doses of IFN-α1 or IFN-α/ApoA1 could protect mice from EAE. To this end, high-dose AAVs (5 × 1011 viral genomes [vg]/mouse) were administered 2 d prior to EAE induction (Fig. 1A). All mice receiving control AAV–GFP developed EAE symptoms, whereas those receiving AAV–IFN-α or –IFN-α/ApoA1 showed mild or even no symptoms at all (Fig. 1B, 1C). However, mice treated with AAV–IFN-α died likely because of the hematological toxicity usually associated with the high and sustained doses of this cytokine (24). All mice treated with AAV–IFN-α/ApoA1 survived (Fig. 1D).

FIGURE 1.
FIGURE 1.

Sustained doses of IFN-α or IFN-α/ApoA1 prevent the onset of clinical signs of EAE in mice. C57BL/6 (n = 6 per group) mice were treated with high-dose (5 × 1011 vg) AAVs encoding GFP, IFN-α, or IFN-α/ApoA1 2 d before MOG immunization (A–D), and clinical score (B), body weight (C), and survival (D) were further determined. Decreasing doses of AAV–IFN-α (5 × 1011, 1 × 1011, and 1 × 1010 vg) were administered to C57BL/6 mice (n = 6 per group) 2 d before MOG immunization (E–G), and clinical score (E), body weight (F), and survival (G) were further determined. Data from the clinical score and weight curves are expressed as mean ± SEM and analyzed by extra sum-of-squares F test. Survival is represented by a Kaplan–Meier plot. Log-rank test. ***p < 0.001.

The next step was to investigate whether lower doses of AAV–IFN-α could protect against EAE development without inducing fatal toxicity. Accordingly, decreasing doses of the AAV–IFN-α (5 × 1011, 1 × 1011, and 1 × 1010 vg/mouse) were administered 2 d before MOG immunization. As illustrated by Fig. 1E and 1F, all three tested doses protected mice from developing EAE, but, importantly, all animals survived only when the lowest dose (1 × 1010 vg/mouse) was used (Fig. 1G).

AAVs encoding IFN-α or IFN-α fused to apolipoprotein A-1 protect from ongoing EAE disease

Although the AAV8-mediated transgene expression reaches its peak at 2 wk after administration (27), we decided to evaluate the use of therapeutic and safe doses of AAVs encoding IFN-α or AAV–IFN-α/ApoA1 in mice undergoing EAE. To this end, mice were treated with the lowest AAV–IFN-α dose (1 × 1010 vg/mouse) 4 d after MOG immunization (Fig. 2A). As shown in Fig. 2B and 2C, they all were almost completely protected from EAE. Mice treated with a safe AAV–IFN-α/ApoA1 dose (5 × 1011 vg/mouse) developed EAE, but symptom severity scores were significantly lower than with control AAV–GFP (Fig. 2B, 2C). Interestingly, the clinical course of EAE declined by 2 wk after virus AAV–IFN-α/ApoA1 administration when maximum AAV expression levels are expected. In this experimental setting, the administration of 10,000 U of recombinant mouse IFN-α three times per week does not improve the clinical symptoms (Fig. 2D, 2E).

FIGURE 2.
FIGURE 2.

Therapeutic administration of IFN-α or IFN-α/ApoA1 reduces the onset of clinical signs of EAE in mice. AAVs encoding GFP, IFN-α, or IFN-α/ApoA1 or recombinant mouse IFN-α (rmIFN-α) were administered to C57BL/6 mice (n = 12 per group) at day four after immunization (A–E). The clinical score (B) and body weight (C) of mice receiving AAVs encoding GFP (1 × 1010 vg), IFN-α (1 × 1010 vg), or IFN-α/ApoA1 (5 × 1011 vg) were recorded over time. For evaluation purposes, the clinical score (D) and body weight (E) of mice receiving the above-mentioned doses of AAVs encoding GFP and IFN-α were compared with those administered with 10,000 U of rmIFN-α i.v. three times per week (n = 13 per group). In another set of experiments (F–H), the clinical score (G) and body weight (H) of mice (n = 9 per group) administered with AAVs encoding GFP (1 × 1010 vg), IFN-α (1 × 1010 vg), or IFN-α/ApoA1 (5 × 1011 vg) 13 d after immunization and once the mean of clinical scores reached an average of two (day 13) were determined. Data from the clinical score and weight curves are expressed as mean ± SEM and analyzed by extra sum-of-squares F test. Survival is represented by a Kaplan–Meier plot. Log-rank test. *p < 0.05, **p < 0.01, ***p < 0.001.

We next decided to evaluate the therapeutic efficacy of the viruses once the disease had been fully developed. To this end, similar AAV doses as above were administered when the average symptom severity scores reached ∼2 (day 13) (Fig. 2F). As shown in Fig. 2G and 2H, mice treated with AAV–IFN-α or AAV–IFN-α/ApoA1 displayed a significant sharp decline in the clinical signs of the disease as compared with the control group.

Therapeutic dose of AAV–IFN does not induce hematological or neurologic toxicity

Next, we analyzed the advent of putative adverse effects following a therapeutic dose of 10 × 1010 vg AAV–IFN-α 4 d after EAE induction. On day 52 after immunization, analysis of peripheral blood cells showed no differences regarding platelet and erythrocyte between untreated mice and the AAV–IFN-α–treated mice (Fig. 3A), which is indicative of absent hematologic toxicity of the selected AAV dose. However, circulating leukocytes were reduced in the AAV–IFN-α–treated group, likely reflecting the known upregulation of CD69 expression and the subsequent retention in secondary lymphoid organs induced by IFN-α (28) (Fig. 3A). To analyze the neurologic toxicity, we determined the cytokine-mediated depression using the tail suspension test at day 40 after immunization. No differences in the immobility time were detected in mice treated with AAV–GFP or AAV–IFN-α (Fig. 3B).

FIGURE 3.
FIGURE 3.

Long-term exposure to low-dose IFN-α does not induce hematological or neurologic toxicity. C57BL/6 mice were immunized and treated with AAV–IFN-α 4 d later. (A) WBC, platelets (PLT), and RBC were quantified from peripheral blood at day 52 after immunization (n = 4, mice per group). (B) Immobility time in a 6-min tail suspension test (n = 10 per group). Data are expressed as mean + SD. t test. *p < 0.05.

Activity of sustained low-dose IFN-α on immune cells

Low-dose (1 × 1010 vg/mouse) AAV–IFN-α was safe and effectively controlled the disease progression and allowed detectable circulating levels of IFN-α at 3 wk post–AAV–IFN-α administration (Fig. 4A). In an attempt to figure out the mechanism of action of this vector dose, we first analyzed the surface expression level of Ly6C, a GPI-linked protein expressed by inflammatory monocytes (29) and considered a biomarker of IFN-α activity (30). As shown by Fig. 4B and Supplemental Fig. 1, Ly6C was upregulated in monocyte populations (Ly6G−CD11b+) of wild-type (WT) mice treated with AAV–IFN-α. In EAE mice, Ly6C was also upregulated by AAV–IFN-α but also control AAV–GFP, reflecting the ongoing inflammatory process (Fig. 4B). Next, the percentages of spleen Tregs were found higher in both the WT and the EAE mice treated with AAV–IFN-α compared with the AAV–GFP control group (Fig. 4C, Supplemental Fig. 2). To confirm that IFN-α was the major driver of Treg expansion in the immunized group and that this population responded to the low concentration of IFN-α in circulation, CD4+CD25+ were isolated, and the levels of different IFN-stimulated genes were assessed. The amount of the IFN-stimulated genes transcripts were significantly higher in the group treated with AAV–IFN-α than in the controls, thus confirming the direct activity of IFN-α on Tregs (Fig. 4D). Neutrophils, which have an important role in the pathogenesis of EAE (31), were dramatically expanded in MOG-immunized animals, and AAV–IFN-α administration reduced the disease-induced neutrophil expansion significantly (Fig. 4E, Supplemental Fig. 1).

FIGURE 4.
FIGURE 4.

Characterization of the effect of sustained low-dose IFN-α on immune cells. (A) Serum IFN-α levels from C57BL/6 mice 3 wk post-AAV–GFP (1 × 1010 vg) or –IFN-α (1 × 1010 vg) administration, as determined by ELISA. (B–E) Analysis of monocyte (Ly6C+LyG−CD11b+), Tregs (CD4+CD25+Foxp3+), and neutrophil (Ly6G+Ly6C+CD11b+) populations in spleens from mice administered with AAV–GFP (1 × 1010 vg) or –IFN-α (1 × 1010 vg) 4 d after being immunized (EAE) or not (WT) with MOG. (B, C, and E). Flow cytometry analysis showing the geometric mean (GM) of Ly6C in Ly6G− CD11b+ (B) and the percentage of CD25+ Foxp3+ in CD4+ (C) and Ly6G+Ly6C+ in CD11b+ spleen cells (E). (D) mRNA levels of different IFN-stimulated genes were quantified by quantitative real-time PCR in CD4+CD25+ cells isolated by cell sorting from spleens of EAE mice. Data are expressed as mean + SD. t test. *p < 0.05, ***p < 0.001.

Sustained low-dose IFN-α decreases EAE-immune mediators

Histological analysis was carried out to correlate symptoms with the inflammatory and demyelinating course of the disease, as occurs in MS. Control mice with high EAE scores had lymphocyte infiltrates in the peripheral regions of the spinal cord in the lumbar region, as indicated by the presence of several nuclei within the white matter. Treatment with sustained low-dose AAV–IFN-α decreased the immune cell infiltration (Fig. 5A, 5B). A demyelinating pattern, defined by a less intense staining of the white matter in the periphery, was also observed in the same regions of the spinal cord where the infiltrates were found. As expected, the reduced infiltration achieved by AAV–IFN-α inversely correlated with demyelination, and this parameter returned to baseline levels in mice that received the AAV–IFN-α (Fig. 5C, 5D).

FIGURE 5.
FIGURE 5.

Sustained low-dose IFN-α decreases EAE-immune mediators. AAVs encoding GFP or IFN-α (1 × 1010 vg) were administered to C57BL/6 mice (n = 12 per group) 4 d after MOG immunization. At day 21, histological and mRNA analyses of the spinal cord were performed. (A) A representative H&E staining is shown. (B) Inflammation was quantified by measuring pixel density. (C) A representative image of Luxol Fast Blue (LFB) Staining is shown. (D) Myelin was quantified by measuring image intensity. (E) Quantitative real-time PCR analysis of mRNA levels of different immune-related genes from spinal cord samples. Data are expressed as mean ± SD. t test for (B) and (E); one-way ANOVA followed by Bonferroni posttest for (E). *p < 0.05, **p < 0.01, ***p < 0.001.

To further characterize the immune cells infiltrating the spinal cord, we performed real-time PCR of several immune-related transcripts 14 d after MOG immunization. All the immune-related markers analyzed (CD4, Foxp3, IFN-γ, IL-17A, and F4/80) were upregulated in the spinal cord of mice treated with AAV–GFP, whereas the levels of these markers were significantly reduced in mice treated with AAV–IFN-α (Fig. 5E).

This unspecific anti-inflammatory activity was accompanied by an Ag-specific dampened immune response. Splenocytes from mice treated with AAV–IFN-α restimulated with MOG peptide in vitro showed an upregulated mRNA expression level of immunosuppressive molecules, such as IL-10, IDO, and programmed death 1 (Fig. 6A). In line with the immunosuppressive program activated by restimulation with the MOG peptide, splenocytes showed both reduced proliferation (Fig. 6B) and release of IL-17A and IFN-γ cytokines (Fig. 6C, Supplemental Fig. 3). Moreover, reduced intracellular IL-17A and IFN-γ cytokine levels were also detected in CD4+ cells (Fig. 6D). To demonstrate the reduced encephalitogenic capacity of MOG-specific spleen T cells from AAV–IFN-α–treated mice, we expanded them in the presence of MOG35–55 peptide, recombinant mouse IL-12, and anti-mouse IFN-γ Ab for further infusion into WT mice. The administration of 10 × 106 cells to WT C57BL/6 mice induced clinical symptoms that peaked 10 d after cell infusion. In contrast, mice that received spleen cells from mice treated with AAV–IFN-α only exhibit mild clinical symptoms and no weight loss (Fig. 6E, 6F).

FIGURE 6.
FIGURE 6.

Sustained low-dose IFN-α increases anti-inflammatory markers and reduces proliferation and IL-17A/IFN-γ production in response to MOG restimulation. C57BL/6 mice were immunized and treated with AAV–IFN-α 4 d postimmunization. At day 21, mice were sacrificed, and splenocytes were isolated. (A) mRNA levels of IL10, Ido, and Pd1 were determined by quantitative real-time PCR. (B) A [3H]thymidine incorporation assay was performed in splenocytes incubated with the MOG peptide (50 μg/ml) for 72 h at 37°C. (C) IL-17A and IFN-γ levels were quantified in the supernatant by ELISA (D) or intracellular by flow cytometry in splenocytes incubated with MOG peptide (50 μg/ml) for 72 h at 37°C. Data are expressed as mean ± SD. t test. *p < 0.05, **p < 0.01, ***p < 0.001. Splenocytes incubated for 3 d in the presence of MOG plus recombinant mouse IL-12 and anti-mouse IFN-γ Ab were inoculated to C57BL/6 mice. Clinical score (E) and body weight (F) were determined. Data are expressed as mean ± SEM and analyzed by extra sum-of-squares F test, ***p < 0.001.

Discussion

Cytokines are soluble mediators of the immune response. Tightly controlled release and short half-life in circulation are common characteristics that ensure timely cross-talk among immune cells. Mimicking these biologic properties of cytokines is a major hurdle for the clinical use of recombinant cytokines. The use of recombinant proteins as drugs requires the administration of high doses to reach relevant concentrations at the target site. However, i.m. or s.c. injections produce peaks and valleys in the pharmacokinetic profile. Thus, plasma concentrations fluctuate, often falling outside the therapeutic range with long periods of supra- and infratherapeutic plasma concentrations. These pharmaceutical issues are exemplified by the clinical use of type I IFNs for the treatment of relapsing-remitting MS and have led to the approval of five different IFN-β drugs. Although compelling evidence is lacking, frequent administration of IFN-β has a beneficial effect on annualized relapse rates (32). An alternative to this approach that is also available for patients with MS is pegylated IFN-β. The binding of 20 kDa polyethylene glycol to IFN-β prolonged its half-life in circulation at the expense of reducing the sp. act. A single dose every 2 or 4 wk of this chemically modified IFN-β is as efficient and safe as other IFN drugs that required several administrations per week (33). In this study, we sought to determine the viability of sustained exposure to the IFN-α1 cytokine using the EAE mouse model. It has been shown previously that hydrodynamic administration of a plasmid encoding IFN-β was able to reduce the symptoms for 1 mo (34). The use of an adeno-associated virus allowed us to take this concept to the extreme. After the administration of hepatotropic AAV-8, transduced hepatocytes continuously release the protein encoded by the gene of interest. Plasma levels reach a stable concentration within the first 2 wk postadministration, and this plasma concentration is maintained throughout the lifespan of mice. In our hands, the administration of AAV–IFN-α exerted a potent therapeutic effect that allowed titrating down the virus to a dose of 1 × 1010 vg per mouse. We have previously reported that this dose does not exert an antitumor effect against colon cancer cells implanted in the liver (24) but produced detectable amounts of the cytokine in circulation. These IFN-α levels also induced the expression of IFN-stimulated genes in circulating Tregs and remodeled the immune cells in the spleen, promoting Ly6C expression on monocytes, reducing neutrophils, and expanding Tregs.

Moreover, this low-dose IFN-α decreased the immune cell infiltration and demyelination of the spinal cord and dampened the immune response against the MOG peptide. Finally, the low doses of AAVs used in this study were safe, and we did not detect hematological or neurologic adverse events. Thus, this study illustrates the safety and therapeutic potential of a sustained delivery of low-dose IFN-α cytokine.

The concept of low-dose cytokine has entered into clinical trials thanks to the use of low-dose IL-2 for the treatment of several autoimmune diseases, such as type 1 diabetes and autoimmune hepatitis (35, 36). This study supports the putative translation of the concept of low-dose cytokines to the treatment of relapsing-remitting MS with IFN-β. However, the uncontrolled release of IFN-β/α from a gene therapy vector could lead to unacceptable severe adverse effects. The use of sophisticated inducible promoters or suicide genes could solve these problems but may have clinical development challenges. Thus, to translate this concept to the clinical arena, the fusion of IFN-α to apolipoprotein A-1 could be a promising alternative. Higher doses of AAVs coding for IFN-α/ApoA1 fusion protein were required to achieve a therapeutic effect when compared with the AAV–IFN-α. This tempered activity would be favorable when using other delivery strategies, such as modified mRNA (37, 38) or s.c. administration of the recombinant fusion protein (39). The deleterious peaks and valleys of the pharmacokinetic profile could be minimized by the increased half-life in circulation and consequently reduce the cytotoxic activity of the fusion protein (26).

The sustained release of low doses of IFN-α leads to a significant increase in Tregs defined as Foxp3+ cells within the CD4+ population. The role of IFN-α on Treg development remains controversial. Early reports showed that both exogenous and endogenous type I IFNs promoted effector CD4+ T lymphocytes with low or negative Foxp3 expression and reduced the percentage of CD4+ cells that expressed high levels of Foxp3 (40). However, experiments using mixed bone marrow chimeras between WT and IFN-α/βR knockout mice highlighted a role for type I IFNs in the development of Tregs (41). It is also known that treatment with IFN-β can induce CD4+CD25+Foxp3+ Tregs in some patients with MS (42). Moreover, IFN-α has been previously shown to disarm Treg suppressive function (without affecting Foxp3 expression) by the downregulation of intracellular cAMP level (43). Thus, the long-term functional outcome of the two opposing effects (expansion and deactivation) of IFN-α on Tregs remains to be determined.

In conclusion, sustained low-dose delivery of IFN-α1 or IFN-α/ApoA1 improved symptoms of the EAE by immune cell remodeling. Thus, feasible clinical strategies to translate the concept of low-dose immunotherapy to relapsing-remitting MS may improve the management of the disease.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

The authors acknowledge Paul Miller for English editing and Eva Martínez-Cáceres for helpful comments.

Footnotes

  • This work was supported by a grant from Instituto de Salud Carlos III (PI16/00668) cofinanced by Fondos Feder (to P.B.) and grants from Worldwide Cancer Research (14-1275), the Fundació La Marató TV3 (201319-30), and the Spanish Ministerio de Economía y Competitividad (SAF2016-80535-R) cofinanced by European Development Regional Fund “A Way to Achieve Europe” (to F.L.) and SAF2016-78568-R (to J.J.L. and N.C.). P.B. and F.A. are supported by an Instituto de Salud Carlos III by Miguel Servet II contract (CPII15/00004) and the Sara Borrell Program (CD15/00016), respectively.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AAV
    adeno-associated viral
    EAE
    experimental autoimmune encephalomyelitis
    IFN-α/ApoA1
    fusion of IFN-α1 to apolipoprotein A-1
    MOG
    myelin oligodendrocyte glycoprotein
    MOG35–55
    MOG peptide 35–55
    MS
    multiple sclerosis
    RPLP0
    ribosomal protein large P0
    Treg
    regulatory T cell
    vg
    viral genome
    WT
    wild-type.

  • Received November 5, 2018.
  • Accepted May 31, 2019.

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