Larval Echinococcus multilocularis infection reduces dextran sulphate sodium-induced colitis in mice by attenuating T helper type 1/type 17-mediated immune reactions

Abstract

The tumour-like growth of larval Echinococcus multilocularis tissue (causing alveolar echinococcosis, AE) is directly linked to the nature/orientation of the periparasitic host immune-mediated processes. Parasite-mediated immune suppression is a hallmark triggering infection outcome in both chronic human and murine AE. So far, little is known about secondary systemic immune effects of this pathogen on other concomitant diseases, e.g. endogenous gut inflammation. We examined the influence of E. multilocularis infection on murine dextran sodium sulphate (DSS) -induced colitis. At 3 months after E. multilocularis infection (chronic stage), the mice were challenged with 3% DSS in the drinking water for 5 days plus subsequently with tap water (alone) for another 4 days. After necropsy, fixed tissues/organs were sectioned and stained with haematoxylin & eosin for assessing inflammatory reactions. Cytokine levels were measured by flow cytometry and quantitative RT-PCR. Colitis severity was assessed (by board-certified veterinary pathologists) regarding (i) colon length, (ii) weight loss and (iii) a semi-quantitative score of morphological changes. The histopathological analysis of the colon showed a significant reduction of DSS-induced gut inflammation by concomitant E. multilocularis infection, which correlated with down-regulation of T helper type 1 (Th1)/Th17 T-cell responses in the colon tissue. Echinococcus multilocularis infection markedly reduced the severity of DSS-induced gut inflammation upon down-regulation of Th1/Th17 cytokine expression and attenuation of CD11b⁺ cell activation. In conclusion, E. multilocularis infection remarkably reduces DSS-induced colitis in mice by attenuating Th1/Th17-mediated immune reactions.

Keywords: T helper type 1/type 17 cells; inflammation; parasitic-helminth.

Figure 1

Mice chronically infected with Echinococcus multilocularis are protected from dextran sodium sulphate (DSS) ‐induced colitis. (a) Weight loss relative to the initial body weight. Mean values of n = 15 to n = 18 mice analysed per group are shown with error bars indicating the SD. At 3 months after E. multilocularis infection, experimental colitis was induced by administration of 3% DSS in the drinking water for 5 days followed by 4 days of regular tap water. Day 1, 2, 3, 4 indicates the days after tap water. (b) Disease activity index (DAI) changes among groups after 9 days 3% DSS treatment at the end time‐point. Data represents the mean ± SD (n = 15 to n = 18). (c) Colon lengths were determined in individual mice. Data show mean values for each group of mice. (d–g) Individual parameters of histopathological scoring. (d) Lymph follicles in the colon. (e) Increase in granulopoiesis in the bone marrow. (f) Lymphoid structures in the spleen. (g) Extramedullary haematopoiesis in the spleen. Histopathological scores were determined for individual mice by a pathologist according to parameters defined in the Materials and methods section. Columns show mean values for n = 5 or n = 6 mice analysed per group and error bars indicate the SD. One representative experiment out of three independent experiments is shown. HPF, high‐power field; EMH, extramedullary haematopoiesis. *P < 0·05.

Figure 2

Representative histopathological findings in the colon [haematoxylin & eosin (H&E) staining]. Representative histopathological changes of the colon [H&E stain, magnification 100× (first row) and 400× (second row)]. Distension of mucosa and submucosa by infiltration of inflammatory cells, fibrosis, loss and proliferation of crypts in dextran sodium sulphate (DSS) group compared with a regular colon without pathological changes in NC, Em and Em+DSS groups. Higher magnification reveals ulceration of the mucosal epithelium with infiltration of neutrophils (asterisk) and lymphocytes in the depth of the lamina propria mucosae (arrow). [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

Colonic inflammation was attenuated in mice with alveolar echinococcosis (AE) regarding dextran sodium sulphate (DSS‐induced colitis. Inflammatory/inhibitory cytokine gene expression levels in the colon of mice (measured by quantitative RT‐PCR). AU, arbitrary units. β‐Actin was used as housekeeping gene. Data represent mean ± SD of three independent experiments of a total of 15–18 mice in each group (five or six mice per group in each independent experiment). Comparison between groups was performed using a one‐way analysis of variance with Bonferroni's multiple comparison post‐test for statistical analysis. *P < 0·004.

Figure 4

Dextran sodium sulphate (DSS) ‐induced colitis changed the splenic immune response in mice with alveolar echinococcosis (AE). Cells were firstly gated on lymphocytes by size and singularity followed by DAPI exclusion to identify live cells for further analysis. Live lymphocytes were gated on CD4 expression to first identify CD4⁺ T cells. Then phycoerythrin (PE) ‐labelled Foxp3, interleukin‐10 (IL‐10), IL‐4 and interferon‐γ (IFN‐γ) expression was identified as Foxp3⁺, IL‐10⁺, IL‐4⁺ and IFN‐γ ⁺ T cells within CD4⁺ T cells. (a) Frequency of Foxp3⁺ T cells within CD4⁺ T cells in spleen cells from each group. (b) Frequency of CD4⁺ IL‐10⁺ T cells in spleen cells from each group. (c) Frequency of IL‐4⁺ T cells within CD4⁺ T cells in spleen cells from each group. (d) Frequency of IFN‐γ ⁺ T cells within CD4⁺ T cells in spleen cells from each group. The corresponding isotype control antibodies were identically labelled as staining controls. Data represent mean ± SD of three independent experiments of a total of 15–18 mice in each group (five or six mice per group in each independent experiment). (e) Representative images of Foxp3⁺, IL‐10⁺, IL‐4⁺, IFN‐γ ⁺ T cells within CD4⁺ T cells in spleen cells from each group. Comparison between groups was performed using a one‐way analysis of variance with Bonferroni's multiple comparison post‐test for statistical analysis. *P < 0·006. NS, no significance.

Figure 5

Antigen‐presenting cell (APC) activation was attenuated in the spleen of mice with alveolar echinococcosis (AE) upon dextran sodium sulphate (DSS) ‐induced colitis. Cells were first gated on size and singularity followed by DAPI exclusion to identify live cells for further analysis. Live cells were gated on CD11b or CD11c expression to first identify CD11b⁺ and CD11c⁺ cells. Then phycoerythrin‐labelled CD80, CD86 expression was identified as CD80, CD86 frequency within CD11c⁺ or CD11c⁺ cells. (a) CD80 frequency within CD11c⁺ and CD11b⁺ cells, CD80 mean fluorescence intensity (MFI) within CD11b⁺ cells in spleen cells from each group. (b) Representative images of CD80⁺ within CD11b⁺ cells in spleen cells from each group. (c) CD86 frequency within CD11c⁺ and CD11b⁺ cells, CD86 MFI within CD11b⁺ cells in spleen cells from each group. (d) Representative images of CD86⁺ within CD11b⁺ cells in spleen cells from each group. Comparison between groups was performed using a one‐way analysis of variance with Bonferroni's multiple comparison post‐test for statistical analysis. *P < 0·004.

Figure 6

Co‐stimulation was attenuated in the spleen of mice with alveolar echinococcosis (AE) upon dextran sodium sulphate (DSS) ‐induced colitis. Cells were first gated on size and singularity followed by DAPI exclusion to identify live cells for further analysis. Live cells were gated on CD11b or CD11c expression to first identify CD11b⁺ and CD11c⁺ cells. Then phycoerythrin‐labelled CD40 expression was identified as CD40 frequency within CD11c⁺ or CD11c⁺ cells. (a) CD40 frequency within CD11c⁺ and CD11b⁺ cells, CD40 mean fluorescence intensity (MFI) in CD11b⁺ cells in spleen cells from each group. (b) Representative images of CD40⁺ within CD11b⁺ cells in spleen cells from each group. Comparison between groups was performed using a one‐way analysis of variance with Bonferroni's multiple comparison post‐test for statistical analysis. *P < 0·004.