Low-dose interleukin-2 alleviates dextran sodium sulfate-induced colitis in mice by recovering intestinal integrity and inhibiting AKT-dependent pathways

Abstract

Several phase 1/2 clinical trials showed that low-dose interleukin-2 (IL-2) treatment is a safe and effective strategy for the treatment of chronic graft-versus-host disease, hepatitis C virus-induced vasculitis, and type 1 diabetes. Ulcerative colitis (UC) is a chronic inflammatory condition of the colon that lacks satisfactory treatment. In this study, we aimed to determine the effects of low-dose IL-2 as a therapeutic for UC on dextran sulfate sodium (DSS)-induced colitis. Methods: Mice with DSS-induced colitis were intraperitoneally injected with low-dose IL-2. Survival, body weight, disease activity index, colon length, histopathological score, myeloperoxidase activity and inflammatory cytokine levels as well as intestinal barrier integrity were examined. Differential gene expression after low-dose IL-2 treatment was analyzed by RNA-sequencing. Results: Low-dose IL-2 significantly improved the symptoms of DSS-induced colitis in mice and attenuated pro-inflammatory cytokine production and immune cell infiltration. The most effective dose range of IL-2 was 16K-32K IU/day. Importantly, low-dose IL-2 was effective in ameliorating the disruption of epithelial barrier integrity in DSS-induced colitis tissues by restoring tight junction proteins and mucin production and suppressing apoptosis. The colon tissue of DSS-induced mice exposed to low-dose IL-2 mimic gene expression patterns in the colons of control mice. Furthermore, we identified the crucial role of the PI3K-AKT pathway in exerting the therapeutic effect of low-dose IL-2. Conclusions: The results of our study suggest that low-dose IL-2 has therapeutic effects on DSS-induced colitis and potential clinical value in treating UC.

Keywords: PI3K-AKT pathway; RNA-sequencing; dextran sulfate sodium; interleukin-2; ulcerative colitis.

Figure 1

Low-dose IL-2 ameliorates clinical signs in mice with DSS-induced colitis. The groups of mice used in the study were Control (n = 25), DSS+PBS (n = 40), DSS+IL-2 (16K IU, n = 30), and DSS+ IL-2 (32K IU, n = 28). (A) Schematic diagram of the experimental design. (B) Kaplan-Meier survival analysis. (C) Percentage body weight change. (D) Disease activity index (DAI), scored from body weight loss, stool, and bleeding. Data are presented as mean values of replicates ± SEM. ***p < 0.001, **p < 0.01, and *p < 0.05 using one-way ANOVA with a post hoc analysis and t-test.

Figure 2

Effect of low-dose IL-2 on the histopathology of DSS-induced colitis. (A) Representative photographs of the colon (upper) and changes in colon length (bottom) (n = 10 for each group). (B) Histopathological analysis in colon tissue by H&E staining. Scale bar, black 500 µm and blue 100 µm. (C) Histopathological changes scored for colon tissue (n = 10 for each group). (D) Myeloperoxidase (MPO) activity of colon tissue. Data are presented as mean values of replicates ± SEM. ***p < 0.001, **p < 0.01, and *p < 0.05 according to t-test.

Figure 3

Low-dose IL-2 suppressed pro-inflammatory markers in the colons of mice with DSS-induced colitis. (A) The relative expression levels of inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-17A, and IFN-γ) and inflammatory enzymes (COX-2 and iNOS) in colon tissue were assessed by qPCR. (n ≥ 5 for each group). (B) ELISA quantification of TNF-α, IL-6, IL-1β, and IL-17 concentration in the supernatant of colon tissue cultured for 1 day (n ≥ 5 for each group). (C) Immunohistochemistry of pro-inflammatory cytokines TNF-α, IL-17, and IFN-γ, myeloid cells (CD11b), and macrophages (F4/80). Red-brown color indicates the protein expression of TNF-α, IL-17, IFN-γ, CD11b, and F4/80. The tissue was counterstained with hematoxylin. Scale bar, 100 µm. Data are presented as mean values of replicates ± SEM. ***p < 0.001, **p < 0.01, and *p < 0.05 according to t-test.

Figure 4

Low-dose IL-2 restored the functions of goblet cells and defects of the intestinal barrier. (A) To detect mucin, colon tissue was stained with AB-PAS staining. Scale bar, black 500 µm and blue 100 µm. (B) The mRNA expression level of MUC2 in colon tissue was determined by qPCR; n = 5 per group. (C) Mucin (MUC2) protein expression in colon tissue was detected by immunohistochemistry. Scale bar, 100 μm. (D) The relative expression levels of tight junction markers (ZO-1 and Occludin) in colon tissue were assessed by qPCR; n = 5 per group. (E) Immunofluorescent staining of Control, DSS+PBS, and DSS+IL-2 with tight junction (ZO-1, claudin-1) and cell-cell adhesion markers (ECAD). Nuclei were stained with DAPI (blue). Scale bar, 100 µm. (F) Immunohistochemistry of intestinal epithelial apoptosis marker (cleaved caspase-3, left). Scale bar 100 µm. Quantification of DAB-stained area with Fuji ImageJ (n = 3 for each group). Data are presented as mean values of replicates ± SEM. ***p < 0.001, **p < 0.01, and *p < 0.05 according to t-test.

Figure 5

Transcriptomic analysis of mice with DSS-induced colitis exposed to low-dose IL-2. (A) The workflow for differential expression analysis based on the RNA-seq data. (B) Principal component analysis (PCA) was performed with the top 500 highest expressed genes for each sample. Control and DSS+IL-2 samples are closely clustered. (n = 3 for each group). (C) A correlation plot was used to cluster samples and generate a heatmap. Dark blue indicates the highest level of similarity between samples. Control and DSS+IL-2 samples showed a highly positive correlation. (D) Hierarchical clustering of 3,393 overlapping genes was performed using the hclust function and the ward.D2 method (left). Red indicates upregulated genes and green indicates downregulated genes. The representative pathway of genes belonging to each cluster is indicated by a bar graph, according to the p-value.

Figure 6

Low-dose IL-2 relieves experimental colitis by regulating the PI3K/AKT and NF-κB pathways. (A) Heatmap of genes known to be involved in the PI3K/AKT pathway from RNA-seq data. Red indicates upregulated genes, while green indicates downregulated genes. (B) qPCR analysis of genes involved in the PI3K/AKT pathway in colon tissue (n = 3 for each group). (C) Representative western blot analysis of PI3K p85, phospho-AKT (Ser473), and AKT levels in colon tissue. (D) Immunofluorescent staining of Control, DSS+PBS, and DSS+IL-2 (16K IU/day) with p-AKT and Ly6G. Scale bar, white 50 µm and yellow 25 µm. (E) qPCR analysis of the expression of NF-κB p65 (n = 3 for each group). (F) Representative western blot analysis of phospho-NF-κB p65 (Ser536) and NF-κB p65. (G) Immunofluorescent staining with iNOS. Nuclei were stained with DAPI (blue). Scale bar, 100 µm. Data are presented as mean values of replicates ± SEM. ***p < 0.001 and **p < 0.01 according to t-test.