Schistosoma mansoni Infection Is Impacted by Malnutrition

Abstract

Schistosomiasis remains one of the most important neglected tropical diseases in the world. It mainly affects developing countries, where it often coexists with malnutrition. Despite this, few studies have investigated the relationship between schistosomiasis and malnutrition. Herein, we evaluate the impact of malnutrition on experimental S. mansoni infection. Mice were divided into 5 groups: Control (Ctrl) diet (14% protein and 10% lipids), low-protein 3% (LP 3%), low-protein 8% (LP 8%), low-fat 2.5% (LF 2.5%), and low-fat 5% (LF 5%). Mice were fed with their respective diets and were infected when a difference of approximately 20% in the body weight between mice from any experimental group and mice from the control group was achieved. Nutritional, parasitological, and immunological parameters were assessed either just before infection and/or approximately 50 days later before mice were perfused. Our results showed that the 3% low-protein diet was the only one capable of establishing malnutrition in mice. Mice fed with this diet showed: (i) significant reduction in body weight and serum albumin levels before infection, (ii) decreased levels of all biochemical parameters evaluated before perfusion, (iii) decreased numbers of schistosome eggs trapped in intestines and impaired parasite fecundity, (iv) a delay in the granuloma development with a smaller granuloma area, and (v) reduced levels of IL-4 and IFN-γ in the liver. Our findings demonstrate that low protein supply leads to malnutrition in mice and impacts the cytokine milieu in the liver and granuloma formation. Additionally, the establishment of our murine malnutrition model will enable future studies aiming to better understand the complex relationships between nutrition, immune responses, and infection outcome.

Keywords: S. mansoni; low-fat diet; low-protein diet; malnutrition; schistosomiasis.

FIGURE 1

Alterations in the body weight of mice fed with different experimental diets. Mice received different restricted diets: Control (Ctrl), Low protein 3 and 8% (LP 3% and LP 8%, respectively) and Low fat 2.5 and 5% (LF 2.5% and LF 5%, respectively). Body weights (A) were measured three times a week. Dotted line indicates the time point when the mice were infected with ∼100 cercariae of S. mansoni. Food (B) and protein intake (C) were measured daily, and are represented as per day/per mouse in grams. Values are presented as mean ± SD in each graph. Results are representative of two independent experiments (n = 12 mice per group). Significant differences were determined by Two-way ANOVA followed by Tukey’s multiple comparison test (A) or one-way ANOVA followed by Holm-Sidak’s (B,C) (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

FIGURE 2

Serum concentrations of albumin, hemoglobin and total protein in mice fed with different experimental diets. Mice received different restricted diets: Control (Ctrl), Low protein 3 and 8% (LP 3% and LP 8%, respectively) and Low fat 2.5 and 5% (LF 2.5% and LF 5%, respectively). Albumin, hemoglobin and total protein levels (from top to bottom) were measured on the first day of the experiment (day 0), before infection with ∼100 cercariae of S. mansoni (∼day 50) and before perfusion of the hepatic portal system (∼day 100). The median is shown on each graph. Results are representative of two independent experiments (n = 10–12 mice per group). Significant differences were determined by Kruskal-Wallis test followed by Dunn’s multiple comparison test. “*” denote differences between LP 3% and Ctrl groups (*p ≤ 0.05; **p ≤ 0.01); “a” denote differences between before infection and before perfusion in each group.

FIGURE 3

Parasitological parameters of mice fed with different experimental diets. Mice received different restricted diets: Control (Ctrl), Low protein 3 and 8% (LP 3% and LP 8%, respectively) and Low fat 2.5 and 5% (LF 2.5% and LF 5%, respectively). Adult worms were recovered approximately 50 days post-infection (A). The numbers of eggs per gram of liver (B) and intestine (C) were determined by optical microscopy after digestion of both tissues with 10% KOH. Fecundity was determined by the ratio between the number of eggs per gram of intestine and the number S. mansoni couples recovered by perfusion (D). Bars represent the median number of worms recovered or eggs per gram of organ. Results are representative of two independent experiments (n = 10–12 mice per group). Significant differences were determined by Kruskal-Wallis test followed by Dunn’s multiple comparison test (***p ≤ 0.001; ****p ≤ 0.0001).

FIGURE 4

Hepatic granuloma area in mice fed with different experimental diets. Mice received different restricted diets: Control (Ctrl), Low protein 3 and 8% (LP 3% and LP 8%, respectively) and Low fat 2.5 and 5% (LF 2.5% and LF 5%, respectively). At 50 days post-infection, sections of liver from each mouse were obtained and slides were prepared and stained with hematoxyline-eosine. Approximately 100 granulomas containing a single well-defined egg at the exudative-productive stage were randomly selected and measured from the livers of mice from each experimental group. The total area of the granulomas was expressed in square micrometers (μm²) and represented as the median (A). Representative histological sections of liver granulomas from each experimental group (B). Frequencies of granulomas in Ctrl and LP 3% groups (C) and representative types of granulomas: E, exudative; EP, exudative-productive; P, productive (D). Percentage of S. mansoni eggs in different stages of development in the ileum: immature (first, second, third or fourth stages), mature and dead (E). Results are representative of two independent experiments (n = 10–12 mice per group). Significant differences were determined by Kruskal-Wallis test followed by Dunn’s multiple comparison test (A), Student’s t-tests (C) or Two-way ANOVA followed by Sidak’s multiple comparison test (E) (*p ≤ 0.05; ****p ≤ 0.0001). Scale bar = 100 μm (100x).

FIGURE 5

Cytokine profile of mice fed with different experimental diets. Mice received different restricted diets: Control (Ctrl), Low protein 3 and 8% (LP 3% and LP 8%, respectively). Serum samples were obtained before infection (∼day 50), and before perfusion (∼day 100) for cytokine measurement. Levels of IFN-γ (A), TNF (B), IL-6 (C), IL-2 (D), IL-4 (E), IL-10 (F), and IL-17 (G) production were measured using the CBA Th1/Th2/Th17 kit. BI: before infection; BP: before perfusion. Results are representative of two independent experiments (n = 6–11 mice per group) and significant differences were determined by one-way ANOVA followed by Holm-Sidak’s multiple comparison test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

FIGURE 6

Cytokine profile in the liver of mice fed with different experimental diets. Mice received Control (Ctrl) or Low protein 3% (LP 3%) diet and liver homogenates were obtained 50 days after infection (before perfusion) for cytokine measurement. Levels of IFN-γ (A), TNF (B), IL-6 (C), IL-2 (D), IL-4 (E), IL-10 (F), and IL-17 (G) were measured using the CBA Th1/Th2/Th17 kit, n = 8–11 mice per group. Significant differences were determined by Student’s t-tests or Mann-Whitney test (*p ≤ 0.05; **p ≤ 0.01).