Echinococcus granulosus sensu stricto and antigen B may decrease inflammatory bowel disease through regulation of M1/2 polarization

Abstract

Background: Inflammatory bowel disease (IBD) is a chronic idiopathic disease characterized by inflammation-related epithelial barrier damage in the intestinal tract. Helminth infection reduces autoimmune disease symptoms through regulation of inflammatory responses based on hygiene theory. However, the underlying mechanisms remain unclear.

Methods: BALB/c mice were infected with microcysts of E. granulosus sensu stricto and drank water containing 3.5% dextran sodium sulfate (DSS) at the 100th day post-infection. After 7 days of drinking DSS, the mouse body weight change and disease activity index (DAI) were recorded every day, and colon length and histological score were evaluated after sacrifice. After injection with antigen B (AgB), inducible nitric oxide synthase (iNOS) and Fizz1 expression and F4/80⁺CD11c⁺ M1 and F4/80⁺CD206⁺ M2 in the peritoneal cells and colon tissues were analysed by qPCR and flow cytometry, respectively. Gut microbiota were profiled by 16S rRNA sequencing of the mouse faecal samples. For in vitro assay, RAW264.7 macrophages were cultured in medium containing AgB before induction by lipopolysaccharide (LPS). Then, NO in the supernatant was measured, and the expression of cytokine genes associated with macrophages were determined by qRT-PCR.

Results: Echinococcus granulosus s.s. infection and AgB significantly reduced the symptoms and histological scores of IBD induced by DSS (P < 0.05). Flow cytometry showed that AgB inoculation increased F4/80⁺ and CD206⁺ in peritoneal cells. The results of qPCR showed that AgB significantly decreased iNOS and increased Fizz1 expression in the colon of mice inoculated by DSS (P < 0.05). Furthermore, AgB injection led to significant changes in the profiles of five genera (Paraprevotella, Odoribacter, Clostridium cluster XlVa, Oscillibacter, and Flavonifractor) in faecal samples. In vitro analysis showed that AgB reduced NO levels (P < 0.01), with a significant decrease in iNOS expression (P < 0.05) in RAW264.7 cells induced by LPS.

Conclusions: Echinococcus granulosus infection and AgB may improve IBD conditions by inducing an M2-predominant cellular (F4/80⁺ CD206⁺) profile and decreasing type 1 macrophages (F4/80⁺CD11c⁺) in the intestinal lamina propria. In addition, AgB intervention induced changes in the microbiota condition of the gastrointestinal duct and reversed NO expression. Thus, AgB may be a drug candidate for IBD treatment.

Keywords: Antigen B; Echinococcus granulosus; Inflammatory bowel disease (IBD); Macrophage polarization.

Fig. 1

Mice were randomly divided into four experimental groups (n = 10 per group). A Experimental plan for testing E. granulosus s.s. infection and IBD. B Experimental plan for determining the effect of AgB against IBD

Fig. 2

Infection with E. granulosus s.s. ameliorated the clinical symptoms of DSS-induced colitis. Course of ulcerative colitis in E. granulosus s.s.-infected and uninfected mice following 7 days of DSS treatment. A Body weight change. Data represent the mean ± SD (n = 7–8 mice in each group). B DAI score (n = 8–10, mean ± SD). DAI score is used as an index of inflammation in the DSS-induced colitis model. In the DSS-alone group, the DAI score was significantly higher (10.63 ± 1.41, P < 0.05) than in the Eg+DSS group (5.45 ± 2.24). C Photograph of gross pathology of colons from different groups of mice. D Colon lengths from mice infected and uninfected with DSS-induced colitis (n = 7–9). Bars represent the mean ± SD for six or more mice per group. These experiments were repeated twice independently (∗ P < 0.05, ∗ ∗ P < 0.01), and data were analysed by one-way ANOVA and unpaired t-test

Fig. 3

Infection with E. granulosus s.s. reduced the pathological score of the colon shown by H&E staining (×200). A Colon tissue histology shown by H&E staining indicating colonic inflammation in the different groups: red star, inflammatory cells infiltrated in the mucosa; dark arrow: crypt abscess. B Comparison of histological scores for colon cellular infiltration and tissue alterations between the DSS and Eg+DSS groups. In the Eg+DSS group, the histological score was significantly decreased (2 ± 0.52, P < 0.05) compared with the DSS group (4 ± 0.53). Unpaired t-test was used in data analysis (6–8 mice per group, ∗ P < 0.05, ∗ ∗ P < 0.01.). These experiments were repeated twice independently

Fig. 4

Colonic inflammation was attenuated in the AgB-treated/DSS-induced colitis model. Purified AgB was injected into mice intraperitoneally before DSS administration. The differences between the DSS group and AgB+DSS group are shown: A Body weight. Bars represent the mean ± SD for 7–10 mice per group, at day 7 post-treatment with DSS. The average body weight of the PBS+DSS group was reduced by 14.27 ± 3.21% (P < 0.05) compared with a reduction in the AgB+DSS group of 4.68 ± 8.24% (P < 0.05). B Length of colon. Bars represent the mean ± SD for nine mice per group. In the PBS+DSS group, the length of the colon was significantly shortened (5.93 ± 0.13 cm, P < 0.05) compared with the AgB+DSS group (6.42 ± 0.16 cm). C DAI scores. In the PBS+DSS group, the DAI score was significantly increased (8.86 ± 1.575, P < 0.05) compared with the AgB+DSS group (6 ± 1). Bars represent the mean ± SEM for seven mice per group. D Colon tissue histology stained with H&E (8–10 mice per group, and the experiment was repeated independently) (×200)

Fig. 5

Cytoflow cytometric analysis of cells isolated from the peritoneal cavity of mice injected with AgB and challenged with DSS. A Cells were first gated on size and singularity followed by DAPI exclusion to identify live cells for further analysis. Live cells were gated on CD45 and F4/80 double expression to identify macrophages. Then, FITC-labelled CD11 expression was identified in M1 cells. B The frequency of macrophage marker CD45 and F4/80 double-positive (25.76 ± 4.97) was higher in the PBS+DSS group than in the other groups (n = 6–8). Bars represent the mean ± SD. C The proportion of type 1 macrophages (F4/80⁺CD11c⁺) was significantly decreased in the AgB group (37.46 ± 15.41) and AgB+DSS group (45.4 ± 17.30) compared with the PBS+DSS (87.99 ± 4.13) and NT groups (90.63 ± 6.68). Bars represent the mean ± SD for 7–10 mice per group. Comparison between groups was performed using one-way analysis of variance (ANOVA) with Bonferroni multiple comparison post-test for statistical analysis (the experiment was repeated independently,   ∗ P < 0.01, ∗ ∗ ∗ P < 0.005)

Fig. 6

Injection with AgB altered the immune status of mice with DSS-induced colitis. LPMCs were collected from mouse intestines and tested by flow cytometry. Live cells were gated on CD45 and F4/80 double expression to identify macrophages; then, FITC-labelled CD11 expression was used to identify M1 cells, and APC-labelled CD206 expression was used to identify M2 cells. Comparison between groups was performed to determine the following: A There was no significant difference in the percentage of macrophages between the PBS+DSS group and other groups (n = 6–10). B The percentage of M1 cells between the groups also showed no significant difference (n = 6–10). C The percentage of M2 cells in the PBS+DSS group was reduced by 11.49 ± 2.95% compared with that in the AgB+DSS group (44.28 ± 6.04%, n = 6–10, P < 0.05). D The ratio of M1/M2 cells. Comparison between groups was performed using one-way analysis of variance (ANOVA) with Bonferroni multiple comparison post-test for statistical analysis (the experiment was repeated independently (n = 6–10). Bars represent the mean ± SD, ∗ P < 0.05, ∗ ∗ P < 0.01, ∗ ∗ ∗ ∗ P < 0.0001)

Fig. 7

AgB intervention in mice altered the macrophage-specific gene expression of DSS-induced colitis. Expression of the transcripts was normalized, and fold change was calculated using the ΔΔCt method against mean control ΔCt; data are representative of three independent experiments. The relative expression of iNOS in the AgB+DSS group decreased significantly by 1.126 ± 0.294 compared with the PBS+DSS group (2.932 ± 1.026, n = 5–6, P < 0.05) (A). The relative expression of Fizz1 in the colon of the AgB+DSS group increased significantly (0.467 ± 0.063) compared with the PBS+DSS group (0.239 ± 0.066, n = 5, P < 0.05) (B)

Fig. 8

AgB influenced the gut microbiota of mice with DSS-induced colitis. Gut microbiota comparisons were made between the AgB+DSS and PBS+DSS groups at the family level (A) and genus level (B). There were five mice in each group. Gastrointestinal microbes were assessed using analysis of molecular variance (AMOVA) in Mothur

Fig. 9

AgB intervention ameliorated the damage due to macrophages in vitro. A RAW264.7 cells were used to test the effect of AgB on macrophage differentiation (×200). B Macrophages were incubated with 1000 ng/ml AgB, and after 1 h, the cells were stimulated by the addition of LPS/IFN-γ. After 20 h, the amount of NO present in the cultured cell supernatant was measured using the nitric acid reduction method. In the LPS+AgB group, the level of NO was significantly decreased (178.11 ± 22.63) compared with the LPS group (243.18 ± 16.45). Comparison between the groups was performed using one-way ANOVA for statistical analysis (the experiment was repeated independently, n = 5, ∗ ∗ P < 0.01)