Therapeutic Effect of IL-21 Blockage by Gene Therapy in Experimental Autoimmune Encephalomyelitis

Abstract

The pathogenic role of the interleukin 21 (IL-21) in different autoimmune diseases, such as multiple sclerosis (MS), has been extensively studied. However, its pleiotropic nature makes it a cytokine that may exhibit different activity depending on the immunological stage of the disease. In this study, we developed a gene therapy strategy to block the interaction between IL-21 and its receptor (IL-21R) by using adeno-associated vectors (AAV) encoding a new soluble cytokine receptor (sIL21R) protein. We tested this strategy in a murine model of experimental autoimmune encephalomyelitis (EAE), obtaining different clinical effects depending on the time at which the treatment was applied. Although the administration of the treatment during the development of the immune response was counterproductive, the preventive administration of the therapeutic vectors showed a protective effect by reducing the number of animals that developed the disease, as well as an improvement at the histopathological level and a modification of the immunological profile of the animals treated with the AAV8.sIL21R. The beneficial effect of the treatment was also observed when inducing the expression of the therapeutic molecule once the first neurological signs were established in a therapeutic approach with a doxycyline (Dox)-inducible expression system. All these clinical results highlight the pleiotropicity of this cytokine in the different clinical stages and its key role in the EAE immunopathogenesis.

Keywords: AAV; EAE; IL-21; Multiple sclerosis; Soluble receptor.

Fig. 1

Biochemical and functional characterization of the sIL21R. a Schematic representation of the transmembrane IL-21R and the designed sIL21R (in gray). b Sequence of the therapeutic molecule, that contains the secretion signal and the extracellular region containing the described IL-21 binding site. c Immunocytochemistry with anti-IL-21R antibody (green) reveals cellular distribution of the soluble receptor in HEK293 cells infected with Ad5.sIL21R. Ad5.Null-infected cells were used as a negative control. Cell nuclei were stained with Hoechst (blue). d Secretion of sIL21R into the extracellular medium was detected in the supernatants (SN) of HEK293 cells infected with Ad5.sIL21R. sIL21R was also detected in the cell lysate (CL). Ad5.eGFP-infected HEK293 cells were used as a negative secretion control. Representative Western blot of three independent experiments with primary anti-IL-21R and anti-eGFP antibody. e sIL21R blocked IL-21-Stat3 phoshorylation signalling by inhibiting its binding to IL-21R. Mouse primary splenocytes were incubated for 15 min with 10 ng/mL of IL-21 that had previously been co-incubated with or without sIL21R. Control: unstimulated splenocyte cell lysate. Representative Western blot and quantification of the pStat3/Stat3 ratio from two independent experiments. Data are represented as mean ± SEM. ****p < 0.0001

Fig. 2

The preventive treatment with AAV8.CMV.sIL21R protects against the development of EAE. Mice were immunized with MOG_40-55 and treated with 5·10¹¹ vg/mouse of AAV8.CMV.sIL21R or AAV8.CMV.Null, 21 days prior to the induction of EAE. a Mean daily clinical score for each group. b sIL21R-treated group showed a statistically significant milder clinical course of the disease compared with the Null group. c Average daily weight changes relative to initial weight on the day of EAE induction. The graphs represent the combined results of two independent experiments (Null, n = 18; sIL21R, n = 19). Treatment did not alter the (d) antigen-specific response (MOG_40-55) or the (e) polyclonal response (PHA-L) in splenocytes. The graphs represent the combined results of two independent experiments (Null: n = 16; sIL21R: n = 18). Data are represented as mean ± SEM. *p < 0.05

Fig. 3

Mice treated with AAV8.CMV.sIL21R have a better CNS histopathological status. a Selected images from a representative animal of each group. b Demyelination, c microglia activation, d axonal damage, and e T-cell infiltration was significantly reduced in mice treated with AAV8.sIL21R, while there were no differences in f the GFAP marking. The CD3.⁺ T-cell infiltrate has been delimited with a dotted line. (Null: n = 8; sIL21R: n = 8). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01

Fig. 4

Preventive treatment with AAV8.CMV.sIL21R modifies the immunological profile of mice with EAE. In the chronic phase, sIL21R-treatment (a) induced a peripheral reduction in IL-10 producing CD4 T cells (CD3⁺CD4⁺IL10⁺) and (b) an increase in the levels of TNFα, IL-18 and a trend towards increased IFNγ in the supernatant of splenocytes stimulated with MOG_40-55. (Null, n = 8; sIL21R, n = 9). In the acute phase, sIL21R-treatment (c) increased the mRNA expression of TGFb1, Tbx21 and Gata3, relative to Null group (Null, n = 8; sIL21R, n = 7). d The blockade of IL-21 also produced a peripheral reduction in the frequency of activated CD4 T-cells (CD25⁺Foxp3⁻ in CD3⁺CD4⁺) and e activated NK cells (CD69⁺ in NK1.1⁺CD3⁻) during the acute phase of EAE. However, f sIL21R promoted an increase in the activation of DCs which could be observed in a higher MFI of MHCII in mDCs (CD11b⁺B220⁻CD11c^highCD8⁻) and g in a higher frequency of MHCII⁺ lDCs (CD11b⁺B220⁻CD11c^highCD8⁺MHCII⁺). (Null, n = 10; sIL21R, n = 10). Data are represented as mean ± SEM (a, b, d, e, f, g) or as mean ± SD (c). ^#p < 0.06, *p < 0.05

Fig. 5

The early therapeutic treatment with AAV8.CAG.sIL21R produces a clinical worsening of EAE. Mice were immunized with MOG_40-55 and treated with 10.¹² vg/mouse of AAV8.CAG.sIL21R or AAV8.CAG.Null the day before (day −1) or the day after (day +1) the induction of EAE. a Mean daily clinical score of EAE-incident mice treated at day −1. b Average daily weight change of incident animals treated at day −1 relative to initial weights on the day of EAE induction. c Mean daily clinical score of incident animals treated at day +1. d Average daily weight change of incident animals treated at day +1 relative to initial weights on the day of EAE induction. Data were obtained from a single experiment (Null (day −1): n = 7; sIL21R (day −1): n = 7; Null (day +1): n = 6; sIL21R (day +1): n = 7). Data are represented as mean ± SEM. *p < 0.05

Fig. 6

The administration of AAV vectors close to the induction of EAE produces a reduction in clinical severity. Mice were immunized with MOG_40-55 and treated with 10¹² vg/mouse of AAV8.CAG.Null 21 days before (day −21), the day before (day −1) or the day after (day +1) the induction of EAE. a Mean daily clinical score of EAE-incident mice of all groups. b Null-treated mice at day −1 and day +1 developed a milder disease compared with Null group preventively treated at day −21. Data were obtained from a single experiment (Null (day −21): n = 7; Null (day −1): n = 7; Null (day +1): n = 6). Data are represented as mean ± SEM

Fig. 7

Dox administration does not alter the clinical course and the treatment with AAV8.TetOn.sIL21R improved established symptoms of EAE. Mice were immunized with MOG_40-55, treated with 10¹² vg/mouse of AAV8.TetOn.Null, AAV8.TetOn.eGFP or AAV8.TetOn.sIL21R 14 days before the induction of EAE, and transgene expression was induced by daily i.p. administration of 25 mg/kg doxycycline (Dox) from the day of onset of neurological symptoms. a Mean daily clinical score of EAE mice treated with vehicle or Dox. b Average daily weight change of EAE animals treated with vehicle or Dox relative to weight prior to onset of neurological symptoms. Day 0 is considered the first day of Dox administration. Data were obtained from a single experiment (Vehicle: n = 6; Dox: n = 7). c Representative flow cytometry histogram of eGFP⁺ hepatocytes of the different experimental groups. d Frequency and e MFI of eGFP.⁺ hepatocytes obtained from healthy and EAE mice treated with AAV8.TetOn.eGFP. Baseline values were provided by control animals without vector and without Dox (n = 2). Data were obtained from a single experiment (Vehicle: n = 3; Dox (Healthy: n = 8; EAE: n = 8)). f Mean daily clinical score of mice administered with AAV8.TetOn.Null or AAV8.TetOn.sIL21R vectors and treated with Dox. g Average daily weight change of EAE-incident mice administered with AAV8.TetOn.Null or AAV8.TetOn.sIL21R vectors and treated with Dox relative to weight prior to onset of neurological symptoms. Day 0 was considered the first day of Dox administration. Data were obtained from a single experiment (Null: n = 7; sIL21R: n = 7). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001