Local delivery of adenoviral vectors encoding murine interleukin 10 induces colonic interleukin 10 production and is therapeutic for murine colitis

Abstract

Introduction: Interleukin 10 knockout (IL-10-/-) mice spontaneously develop a Th1 T cell mediated colitis with many similarities to Crohn's disease. Daily injections of IL-10 are unable to induce remission in mice with established disease. In contrast, we have shown previously that intravenous administration of adenoviral vectors encoding IL-10 (AdvmuIL-10) induces hepatic IL-10 release and leads to long term disease suppression with profound systemic immunoregulatory changes.

Aims: To determine whether rectal delivery of AdvmuIL-10 induces localised colonic IL-10 expression without systemic immune suppression, and assess its therapeutic efficacy in IL-10-/- mice with established colitis.

Results: A single rectal infusion of 5 x 10(8) PFU AdvmuIL-10 to 10 week IL- 10-/- mice resulted in a median level of 27.3 pg/mg IL-10 in colonic homogenates harvested one week later. IL-10-/- mice with established colitis treated with an enema of 5 x 10(8) PFU AdvmuIL-10 entered clinical and histological remission whereas empty cassette adenovirus (Adv0) or phosphate buffered saline (PBS) treated mice developed progressive disease. After four weeks, the histological score of AdvmuIL-10 treated mice (4.4 (1.5)) was significantly lower than that of Adv0 (11.1 (1.1); p<0.001) and PBS (10.9 (1.0); p<0.01) treated controls. In addition, the stool concentration of IL-1beta over the four week experiment was significantly higher in mice treated with saline or Adv0 than in those treated with AdvmuIL-10 (p<0.01).

Conclusion: Local AdvmuIL-10 therapy reverses colitis in IL-10-/- mice without the systemic effects seen after intravenous administration. Gene therapy strategies using adenoviral vectors encoding immunoregulatory cytokines may prove to be a potent approach to the treatment of chronic inflammatory diseases such as Crohn's disease.

Figure 1

Adenoviral vector encoding murine interleukin 10 (AdvmuIL-10) induced bioactive IL-10 release from epithelial cells in vitro. HT29 and SW620 cells were plated at 1×10⁶ /ml in a 12 well plate and infected with AdvmuIL-10 or empty cassette adenoviral vector (Adv0, MOI 50:1). Supernatants were sampled at 36 hours and daily thereafter. Cultures were passaged 1:2 every week. No IL-10 was detected in Adv0 infected epithelial cells. (A) IL-10 release from AdvmuIL-10 infected cells. (B) RAW cells (2×10⁵) were plated onto a 96 well plate and incubated with IL-10 10 ng/ml, dilutions of the supernatant (Sup) from AdvmuIL-10 infected HT29 cells, or supernatant that had been preincubated with 10 μg/ml (final) antimurine IL-10 antibody for one hour. Cells were cultured for 24 hours with or without 10 ng/ml lipopolysaccharide (LPS). Supernatants were harvested and assayed for tumour necrosis factor α (TNF-α) release by ELISA. Results are expressed as a percentage of the LPS induced TNF-α response (mean of three experiments). Identical results were obtained using supernatants from SW620 cells.

Figure 2

Local adenoviral vector encoding murine interleukin 10 (AdvmuIL-10) induced colonic IL-10 release. Ten week old IL-10−/− mice received 5×10⁸ PFU of AdvmuIL-10 or saline vehicle either by tail vein injection (A) or rectal infusion (B) under light sedation (n=3/group). After seven days, mice were sacrificed by cervical dislocation. The liver, spleen, and colon were homogenised in 5 μl phosphate buffered saline (PBS) per mg tissue. After centrifugation, supernatants were assayed for murine IL-10 by ELISA (sensitivity 8 pg/mg).

Figure 3

Local adenoviral vector encoding murine interleukin 10 (AdvmuIL-10) was therapeutic for established murine colitis. IL-10−/− mice (n=10/group) with clinical evidence of colitis and C57BL/6×DBA1 wild-type controls (n=5/group) received 5×10⁸ PFU of AdvmuIL-10, empty cassette adenoviral vector (Adv0), or vehicle by rectal instillation under light sedation. (A) Clinical score (one point each for rectal mucous, rectal prolapse, diarrhoea, and weight loss >5% body weight) and (B) stool IL-1β levels were measured weekly, and are expressed as mean (SEM) for each group. **p<0.01, ***p<0.001, two way ANOVA.

Figure 4

Local adenoviral vector encoding murine interleukin 10 (AdvmuIL-10) therapy reduced histological colitis scores in IL-10−/− mice with established disease. Four weeks after therapy, mice were sacrificed by cervical dislocation and sections of five regions of the colon were processed for histology. Representative samples from an IL-10−/− mouse treated with empty cassette adenoviral vector (Adv0) (A) and AdvmuIL-10 (B) are shown. An investigator, blinded to treatment group, gave each section an inflammatory score from 0 to 4. The sum of these scores is shown for each mouse (C), with the bar representing the mean for the group (**p<0.01 versus IL-10−/− saline group; ***p< 0.001 versus IL-10−/− Adv0 group; one way ANOVA with Bonferroni correction).

Figure 5

Rectal adenoviral vector encoding murine interleukin 10 (AdvmuIL-10) did not diminish the lipopolysaccharide (LPS) splenocyte response in IL-10−/− mice. Splenocytes were isolated at sacrifice from both IL-10−/− (n=10/group) and wild-type (n=5/group) mice treated with 5×10⁸ PFU empty cassette adenoviral vector (Adv0), saline, or AdvmuIL-10 by rectal instillation four weeks previously. Cells (2×10⁶ per well) were cultured for 24 hours in the presence of LPS 10 μg/ml. Supernatants were harvested and assayed for tumour necrosis factor α (TNF-α) by ELISA. Cells from each animal were assayed in triplicate; results are expressed as mean (SEM) for each group. *p⩽0.05; **p⩽0.01,*** p<0.001 versus pooled wild-type (WT) mice.

Figure 6

Rectal adenoviral vector encoding murine interleukin 10 (AdvmuIL-10) induced a diminished antiadenoviral response in IL-10−/− mice. The neutralising antiadenovirus antibody titre was analysed in serum from (A) IL-10−/− and (B) wild-type mice four weeks after rectal instillation of 5×10⁸ PFU of AdvmuIL-10, empty cassette adenoviral vector (Adv0), or saline vehicle. Serial serum dilutions were incubated for 90 minutes at 37°C with 2×10⁶ PFU of adenoviral vector encoding β-galactosidase (Advβgal) and applied in duplicate to 80% confluent 293 cells on a 96 well plate. After one hour at 37°C, 50 μl of Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum was added to each well, and cells were cultured for a further 36 hours. Cell supernatants were then removed and assayed using a β-galactosidase substrate solution. In this assay, a low optical density reflects a high titre of serum antiadenoviral antibodies. Results are expressed as mean (SEM) optical density for each group. ***p<0.001 compared with saline treated mice, two way ANOVA).