Therapeutic effect of a new immunosuppressive agent, everolimus, on interleukin-10 gene-deficient mice with colitis

Abstract

A limited number of therapeutic strategies are currently available for patients with inflammatory bowel disease (IBD). In particular, the maintenance therapy after remission in Crohn's disease (CD) is not satisfactory and new approaches are needed. Interleukin-10 gene-deficient (IL-10-/-) mice, a well-characterized experimental model of CD, develop severe chronic colitis due to an aberrant Th1 immune response. Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), a new immunosuppressive reagent, has been used successfully in animal models for heart, liver, lung and kidney transplantation. In the present study, we examined the efficacy of everolimus in the treatment of chronic colitis in an IL-10-/- mouse model. Everolimus was administered orally for a period of 4 weeks to IL-10-/- mice with clinical signs of colitis. The gross and histological appearances of the colon and the numbers, phenotype and cytokine production of lymphocytes were compared with these characteristics in a control group. The 4-week administration of everolimus resulted in a significant decrease in the severity of colitis, together with a significant reduction in the number of CD4+ T cells in the colonic lamina propria as well as IFN-gamma production in colonic lymphocytes. Everolimus treatment of established colitis in IL-10-/- mice ameliorated the colitis, probably as a result of decreasing the number of CD4+ T cells in the colonic mucosa and an associated reduction in IFN-gamma production.

Fig. 1

Clinical and pathologic features of everolimus-treated and untreated control mice. (a) IL-10^–/– mice treated with everolimus (•) gained weight steadily throughout the study period. In contrast, untreated control mice (○) lost weight (significantly different from control weights after 3 weeks). The data represent values (mean ± s.e.m.) from three different experiments (12 mice per group). (b) Grossly, the colon of a mouse treated with everolimus is less thickened and longer than the colon of an untreated mouse. The size of the SP and MLN are smaller in treated mice. (c) Histologically, the colon of an everolimus-treated mouse has less crypt elongation, less inflammatory infiltrate in the lamina propria and more goblet cells than a control mouse. Sections were stained with haematoxylin and eosin. (d) The mean histological score in the colons of treated mice (•, 9·8 ± 1·1) is significantly lower than that in control mice (○, 17·3 ± 3·3). The data represent values (mean ± s.e.m.) (eight mice per group). *P < 0·05 versus control.

Fig. 1

Clinical and pathologic features of everolimus-treated and untreated control mice. (a) IL-10^–/– mice treated with everolimus (•) gained weight steadily throughout the study period. In contrast, untreated control mice (○) lost weight (significantly different from control weights after 3 weeks). The data represent values (mean ± s.e.m.) from three different experiments (12 mice per group). (b) Grossly, the colon of a mouse treated with everolimus is less thickened and longer than the colon of an untreated mouse. The size of the SP and MLN are smaller in treated mice. (c) Histologically, the colon of an everolimus-treated mouse has less crypt elongation, less inflammatory infiltrate in the lamina propria and more goblet cells than a control mouse. Sections were stained with haematoxylin and eosin. (d) The mean histological score in the colons of treated mice (•, 9·8 ± 1·1) is significantly lower than that in control mice (○, 17·3 ± 3·3). The data represent values (mean ± s.e.m.) (eight mice per group). *P < 0·05 versus control.

Fig. 2

(a) Four weeks' treatment with everolimus (▪, n = 10) significantly decreased the number of SP, MLN and CLP compared with those in control (□, n = 10) mice. Mean ± s.e.m. *P < 0·05. (b) Flow cytometric analysis using lymphocytes collected after the 4-week treatment revealed that the numbers of CD3+ and CD4+ T lymphocytes from SP and CLP were significantly diminished. CD8+ lymphocytes from SP and B220+ lymphocytes from CLP were also significantly diminished. □, control (n = 8); ▪, everolimus (n = 8); mean ± s.e.m.; *P < 0·05 versus control.

Fig. 3

(a) Representative results showing the decrease in the number of CD3⁺CD4⁺CD44^high cells of CLP by the daily administration of everolimus for 4 weeks. This is a CD3 gated FACS analysis, CD4⁺CD44^high double positive (RU) rate is decreased (46–24%) by treatment of everolimus. The experiment was repeated three times, and representative results are shown. (b) This figure shows that there is no significant difference between the control group and the treatment group in the number of CD4⁺CD25⁺ T cells gated on CD3⁺ lymphocytes of both the SP and CLP (SP, control 4·6 ± 0·9% versus everolimus 3·1 ± 0·6%; CLP, control 3·4 ± 1·1% versus everolimus 3·3 ± 1·1%; n = 5). (c) This figure shows that each of CD4⁺CD25⁺ regulatory T cells significantly suppressed the proliferation of their responder cells in the co-culture experiment. However, there was no significant difference between the untreated and the treated group in the immunoregulatory function of CD4⁺CD25⁺ T cells (each group n = 4). □, CD4⁺CD25^–. ▪, CD4⁺CD25; mean ± s.e.m. *P < 0·05 versus control.

Fig. 4

INF-γ and IL-4 production by CLP lymphocytes after the daily administration of everolimus for 4 weeks. No difference in the production of IL-4 by CLP lymphocytes was found. However IFN-γ is significantly diminished. The data represent the values (mean ± s.e.m.) (eight mice per group). Closed (▪) and open (□) bars represent everolimus-treated group and the untreated groups, respectively. *P < 0·05 versus control.

Fig. 5

Phosphorylation of eIF4E-binding protein 1 (4E-BP1), a substrate of mTOR kinase, was suppressed in CLP lymphocytes from both wild-type (IL-10^+/+) and IL-10^–/– mice after the daily administration of everolimus for 4 weeks. The three bands that represent the different phosphorylation status are indicated as α, β, γ. The experiment was repeated three times, and representative results are shown.