IL-1β pre-stimulation enhances the therapeutic effects of endometrial regenerative cells on experimental colitis

Abstract

Background: Ulcerative colitis (UC) is a chronic, relapsing, and non-specific inflammatory bowel disease, and the current treatment strategies were mainly used to relieve symptoms or for maintenance. Endometrial regenerative cells (ERCs) are mesenchymal-like stromal cells and have been demonstrated to alleviate multiple immune-dysregulation diseases. Pro-inflammatory stimuli were reported to enhance the immunosuppressive functions of ERCs, but the mechanism underlined is not fully understood. Here, we have designed this study to investigate the therapeutic effects of IL-1β-primed ERCs in the attenuation of experimental colitis.

Methods: BALB/c mice were given 3% dextran sodium sulfate (DSS) for 7 consecutive days and free tap water for 3 days sequentially to induce experimental colitis. PBS (200 μL), ERCs, and IL-1β-primed ERCs (10ng/mL, 48 h) were injected (1 million/mouse/day, i.v.) on day 2, 5, and 8, respectively. Colonic and splenic samples were harvested on day 10 after DSS induction.

Results: It was found that IL-1β-primed ERC treatment markedly attenuated colonic damage, body weight loss, and colon length shortening in colitis mice. Compared with other treatments, cell populations of CD4⁺IL-4⁺Th2 cells, CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Tregs), and CD68⁺CD206⁺ macrophages in spleens were also significantly upregulated in the IL-1β-primed ERC-treated group (p < 0.05). In addition, lower expression of pro-inflammatory (IFN-γ, IL-17, TNF-α, and IL-6), but higher levels of anti-inflammatory cytokines (IL-4 and IL-10) were detected in colons in the IL-1β-primed ERC-treated group (p < 0.05 vs. other groups). Importantly, we also found that different generations of ERCs had an overall lower secretion of Dickkopf-1 (DKK1) by IL-1β pre-stimulation (p < 0.05) and a higher expression of β-catenin in colonic and splenic tissues after the administration of IL-1β-primed ERCs.

Conclusions: This study has demonstrated that IL-1β pre-stimulation effectively downregulated DKK1 expression in ERCs, which in turn promoted the wnt/β-catenin pathway activation in colonic and splenic tissues. Consequently, IL-1β-primed ERCs exhibited an enhanced therapeutic effect in the attenuation of DSS-induced colitis.

Keywords: Colitis; Dickkopf-1; Endometrial regenerative cells; Immunoregulation; Mice.

Fig. 1

Morphology, phenotype, and differentiation potential of ERCs. A The 3rd–5th passage (P3–P5) ERC morphology. B Cell surface markers of ERCs were identified, indicating that ERCs highly expressed CD29, CD44, and CD90, but without expression of CD45. C Differentiation potential of ERCs and IL-β-primed ERCs. a, b Adipocytic differentiation. Red areas are lipid vacuoles which were stained by Oil Red. c, d Osteocytic differentiation. Purple staining indicated the synthesis of alkaline phosphatases by osteoblasts

Fig. 2

IL-1β-primed ERCs alleviate the symptoms of DSS-induced colitis. A Representative pictures showing bloody stool were taken on the 10th day after DSS induction. The mice in the IL-1β-primed ERC group were in the best condition than that in other groups. Body weight changes (B) and Disease Activity Index (DAI) score (C) of each group of mice were recorded daily. In the IL-1β-primed ERC group, the weight loss and DAI score were shown lesser than other groups. D, E The length of the colon in each group was measured and analyzed on the 10th day (n = 6). F Photograph (× 200, H&E staining) of representative histological sections of mouse colons in each group. Arrows indicated the inflammatory cell infiltration. G Histopathological scores were calculated according to the scoring system directed by Singh et al. [31] to assess the colonic injury quantitatively. Data were presented as mean ± standard deviation (SD) (*p < 0.05, **p < 0.01, ***p < 0.001). Statistical analysis was calculated by using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test

Fig. 3

IL-1β-primed ERCs reduced Th1 and Th17, but enhance Th2 and Treg populations in colitis mice. Splenocytes were collected on the 10th day after DSS induction. To accurately identify the subpopulation of Th1, Th2, and Th17 cells, splenocytes were firstly incubated with a cell stimulation cocktail for 5 h before being stained with fluorescent antibodies. A Representative dot plots of CD4⁺IFN-γ⁺ Th1 cells, CD4⁺IL-4⁺ Th2 cells, and CD4⁺IL-17⁺ Th17 cells were shown while positive cells were counted from the quadrant Q2. B Dot plots of CD4⁺CD25⁺Foxp3⁺ Tregs. C Percentage of CD4⁺IFN-γ⁺ Th1 cells. D Percentage of CD4⁺IL-4⁺ Th2 cells. E Percentage of CD4⁺IL-17⁺Th17 cells. F Percentage of CD4⁺CD25⁺Foxp3⁺ Tregs. Data were mean ± SD (n = 6, *p < 0.05, **p < 0.01, ***p < 0.001). p values were calculated by one-way ANOVA followed by the LSD test

Fig. 4

IL-1β-primed ERCs reduced the population of mature DCs, but increased M2 macrophages in colitis mice. To determine whether each treatment has an influence on regulating DC and macrophage phenotypes, anti-CD11c antibody and antigen presenting-related antibodies (anti-MHCII, anti-CD86) were used to measure mature DCs, while anti-CD68 antibody and anti-CD206 antibody were used for M2 phenotype macrophages in spleens. A Representative dot plots of CD11c⁺MHCII⁺ DCs and CD11c⁺CD86⁺ DCs in spleens. B Dot plots of CD68⁺CD206⁺ macrophages. C–E Percentage of CD11c⁺MHCII⁺ DCs, CD11c⁺CD86⁺ DCs, and CD68⁺CD206⁺ macrophages, respectively. Data were mean ± SD (n = 6, *p < 0.05, **p < 0.01, ***p < 0.001). p values were analyzed by one-way ANOVA followed by the LSD test

Fig. 5

IL-1β-primed ERCs reduced macrophage infiltration and inflammatory cytokine expressions in colons. Intra-colon macrophage infiltration was evaluated by immunohistochemical staining. Specifically, we stained iNOS for detecting M1 cell infiltration, and CD206 for measuring M2 cell infiltration. The represent IHC images of mouse colons and quantitive data for cell counts of each group are shown in A (× 200) and B, respectively. The concentrations of inflammatory cytokine productions in colonic tissues were determined by ELISA kit and the relative mRNA expression changes were performed by real-time PCR. IFN-γ (C, D), IL-17 (E, F), TNF-α (G, H), IL-6 (I, J), IL-4 (K, L), and IL-10 (M, N) were shown, respectively, which are majorly secreted by Th1, Th2, Th17 cells, or CD206⁺ macrophages, and closely associated with the development of UC. Data were presented as mean ± standard deviation, and p values were calculated by using one-way ANOVA followed by the least significant difference (LSD) test (n = 6, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 6

IL-1β-primed ERCs decreased DKK1 secretion, but promoted β-catenin expression in splenic and colonic tissues. A The DKK1 level in supernatants of 3rd–7th-generation ERCs were detected by ELISA kit. The 5th generation of ERCs were recorded with the lowest DKK1 expression when compared with other generations (vs. the 5th generation of ERCs, *p < 0.05, **p < 0.01, ***p < 0.001). B DKK1 mRNA expression in P5 ERCs (n = 3). C DKK1 protein expression in P5 ERCs (n = 3). D DKK1 secretion in P5 ERC supernatants (n = 3). E–J The mRNA expression level of β-catenin in spleens (E) and colons (H) was detected, respectively. Furthermore, the protein level of β-catenin in spleens (F) and colons (I) was also demonstrated. Western blot analysis of β-catenin protein expression in the spleen (G) and colon (J) tissue homogenate. Data were presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). Statistical analysis was performed by one-way ANOVA followed by the LSD test

Fig. 7

IL-1β-primed ERCs promoted the anti-inflammatory profile transformation through Wnt/β-catenin pathway activation. Wnt/β-catenin pathway is participating in regulating the development and differentiation of immunocytes and exhibiting anti-inflammatory effects in chronic disease. In the present study, DKK1 expression in ERC turned to be decreased after priming with IL-1β. After infusing IL-1β-primed ERCs into colitis mice, it was found that the β-catenin expression in splenic and colonic tissues were strikingly increased. Moreover, the inflammatory profile (Th1, Th17, M1, IFN-γ, IL-17, TNF-α, and IL-6) was transformed into an anti-inflammatory state (Th2, Treg, M2, IL-4, and IL-10), which is in line with the β-catenin expression changes. Therefore, in this study, we hypothesize that downregulating DKK1 expression in ERCs could promote the wnt/β-catenin pathway activation in immune cells, which would enhance the anti-inflammatory properties and optimize the immunomodulatory effect of ERCs