Schistosome Eggs Impair Protective Th1/Th17 Immune Responses Against Salmonella Infection

Abstract

Countries with a high incidence of helminth infections are characterized by high morbidity and mortality to infections with intracellular pathogens such as Salmonella. Some patients with Salmonella-Schistosoma co-infections develop a so-called "chronic septicemic salmonellosis," with prolonged fever and enlargement of the liver and spleen. These effects are most likely due to the overall immunoregulatory activities of schistosomes such as induction of Tregs, Bregs, alternatively activated macrophages, and degradation of antibodies. However, detailed underlying mechanisms are not very well investigated. Here, we show that intraperitoneal application of live Schistosoma mansoni eggs prior to infection with Salmonella Typhimurium in mice leads to an impairment of IFN-γ and IL-17 responses together with a higher bacterial load compared to Salmonella infection alone. S. mansoni eggs were found in granulomas in the visceral peritoneum attached to the colon. Immunohistological staining revealed IPSE/alpha-1, a glycoprotein secreted from live schistosome eggs, and recruited basophils around the eggs. Noteworthy, IPSE/alpha-1 is known to trigger IL-4 and IL-13 release from basophils which in turn is known to suppress Th1/Th17 responses. Therefore, our data support a mechanism of how schistosomes impair a protective immune response against Salmonella infection and increase our understanding of helminth-bacterial co-infections.

Keywords: IPSE/alpha-1; Salmonella Typhimurium; Schistosoma mansoni; Th1; Th17; co-infection; immunoregulation.

Figure 1

S. mansoni eggs (Sm) impair clearance of Salmonella (STm) infection. Mice were injected intraperitoneally with Sm and 8 days later they were orally infected with STm. Mice were sacrificed on day 1 (A), week 2 (B), and week 5 (C) p.i.. Feces were collected three and four weeks p.i.. (D). Each data point represents one animal and the means + SD are shown (n = 5–10 mice per group). Dashed line indicates detection limit. Data were analyzed using one-way ANOVA with Tukey's post-test (A-C) or Student's t-test (D). ***p < 0.001; **p < 0.01; ns, p ≥ 0.05 (not significant).

Figure 2

S. mansoni eggs (Sm) modulate Salmonella-induced intestinal pathology. Histopathological changes in the lumen, surface epithelium, mucosa, and submucosa were analyzed in H&E-stained sections of the proximal colon. (A) H&E-stained colon sections on day 1 p.i. with STm. (B) H&E-stained colon sections 2 weeks p.i.. (C) Pathology scores of week 2 p.i.. (D) H&E-stained colon sections 5 weeks p.i.. (E) Pathology scores of week 5 p.i.. Each data point represents one animal and means + SD are shown (n = 5–10 mice per group). Scale bars 200 μm for 40X and 100 μm of 100X magnifications. Data were analyzed using one-way ANOVA with Tukey's post-test. ***p < 0.001; **p < 0.01; ns, p ≥ 0.05 (not significant).

Figure 3

Accumulation of neutrophils upon treatment with S. mansoni eggs (Sm) and infection with S. Typhimurium (STm). (A–D) Pathology scores of the different areas at week 5 p.i.. (E) Intestinal tissue sections were stained with antibodies against MPO to visualize neutrophils. Note the accumulation of MPO positive cells in the lumen of Sm+STm-infected mice compared to only a few MPO positive cells in STm-infected mice (upper panels). In contrast, MPO positive cells were found in the mucosa of STm-infected mice but only very few in Sm+STm-infected mice (lower panels). No MPO positive cells were visible in sections from control mice or mice injected with Sm alone. Red, MPO; blue, DAPI. Scale bar 50 μm. Data were analyzed using Student's t-test. **p < 0.01; *p < 0.05; ns,p ≥ 0.05 (not significant).

Figure 4

S. mansoni eggs (Sm) induce granuloma formation in the visceral peritoneum. (A) H&E-stained sections of Sm-induced granulomas in the visceral peritoneum attached to the colon. Scale bars 200 μm for 40X, 100 μm for 100X, and 50 μm for 200X magnifications. (B) Sections of intestinal tissue were stained with antibodies against IPSE/alpha-1 (brown). Scale bars 100 μm (left panel) and 50 μm (right panel). (C) IL-13 producing basophils were found in the granuloma; basophils (mMCP8, green); IL-13 (red); nuclei (DAPI, blue). Scale bar 100 μm. Note the autofluorescence of the Sm shell (green). Second row: magnification of the area indicated by box 1 containing the majority of IL-13 producing basophils. Bottom row: magnification of the area indicated by box 2 containing mainly basophils which do not produce IL-13. (D) H&E stains with necrotizing granuloma containing numerous S. mansoni eggs with central necrosis in the visceral peritoneum (left panel). The granuloma contains numerous eosinophilic granulocytes (middle panel, arrowheads). Chloroacetate esterase (CAE) staining identifies scattered neutrophilic granulocytes in the granuloma (right panel, asterisks) while eosinophilic granulocytes remain negative (right panel, arrowheads). Scale bars are 100 μm in left panel and 10 μm in middle and right panel, respectively.

Figure 5

S. mansoni eggs (Sm) impair the STm-induced IL-17 response. RNA was isolated from intestinal tissue at day 1, week 2, and week 5 p.i.. STm infection upregulated cytokine responses including (A) Ccl2, (B) Ifng, (C) Il13, (D) Tnfa and (E) Il17a at all time points. Sm significantly downregulated expression of Ifng and Ccl2 at day 1 p.i. and Il17a at week 5 p.i. while the other cytokines tested were not affected. Each data point represents one animal and box and whiskers indicating the minimum and maximum values are shown (n = 5–10 mice per group). Data were analyzed using one-way ANOVA with Tukey's post-test. Data analyses are only depicted for comparison of STm with Sm+STm groups. **p < 0.01; *p < 0.05; ns, p ≥ 0.05 (not significant).