Malnutrition alters the innate immune response and increases early visceralization following Leishmania donovani infection

Abstract

Malnutrition is a risk factor for the development of visceral leishmaniasis. However, the immunological basis for this susceptibility is unknown. We have developed a mouse model to study the effect of malnutrition on innate immunity and early visceralization following Leishmania donovani infection. Three deficient diets were studied, including 6, 3, or 1% protein; these diets were also deficient in iron, zinc, and calories. The control diet contained 17% protein, was zinc and iron sufficient, and was provided ab libitum. Three days after infection with L. donovani promastigotes, the total extradermal (lymph nodes, liver, and spleen) and skin parasite burdens were equivalent in the malnourished (3% protein) and control mice, but in the malnourished group, a greater percentage (39.8 and 4.0%, respectively; P = 0.009) of the extradermal parasite burden was contained in the spleen and liver. The comparable levels of parasites in the footpads in the two diet groups and the higher lymph node parasite burdens in the well-nourished mice indicated that the higher visceral parasite burdens in the malnourished mice were not due to a deficit in local parasite killing but to a failure of lymph node barrier function. Lymph node cells from the malnourished, infected mice produced increased levels of prostaglandin E(2) (PGE(2)) and decreased levels of interleukin-10. Inducible nitric oxide synthase activity was significantly lower in the spleen and liver of the malnourished mice. Thus, malnutrition causes a failure of lymph node barrier function after L. donovani infection, which may be related to excessive production of PGE(2) and decreased levels of IL-10 and nitric oxide.

FIG. 1

(A) Growth characteristics of weanling mice fed the four experimental diets A to D for 4 weeks. Mice (n = 10 for each diet group) were changed to the experimental diets shortly after weaning and continued for 28 days. Diet A (control) contained 17% protein, was zinc and iron sufficient, and was provided ad libitum. Diets B, C, and D contained 6, 3, and 1%, protein, respectively, and were zinc, iron, and calorie deficient (see Table 1). Shown are growth curves over 28 days after the change to the experimental diets. The significance of the differences in mean values of percent weight gain on day 28 between the control (diet A) and the malnourished (diets B, C, and D) mice was determined by Student's t test. Data are given as mean and SEM. (B) Effect of malnutrition on early visceralization of L. donovani to the liver and spleen. Mice (n = 6 for each diet group) were fed the four experimental diets A to D described for panel A and were infected in the hind footpad with 5 × 10⁶ metacyclic promastigotes on day 28 after initiation of the experimental diet. The mice were euthanized 3 days after infection, and the parasite burden in the liver and spleen was determined by quantitative limiting-dilution culture. Parasite visceralization was considered to have occurred if parasites were detected in the culture of the lowest dilution of tissue. Percent visceralization is defined as the number of organs (spleen or liver) with detectable parasites divided by the total number of organs. The significance of the differences in the proportion of mice that showed visceralization between the normal control group (diet A) and the malnourished groups (diets B, C, and D) was determined using the chi-square test.

FIG. 2

(A) NO production by resident peritoneal cells from mice in the four diet groups A to D. Resident peritoneal cells were cultured in medium alone or with IFN-γ (20 U/ml) and LPS (1 μg/ml) for 24 h. Supernatants were tested for total nitrate and nitrite by the Greiss reaction. The results are expressed as the mean and standard error of the mean of the nitrate/nitrite concentration from the stimulated and unstimulated cultures (n = four mice per group). Differences in mean values were determined by Student's t test. (B) Splenic and hepatic NOS enzyme activity in control (diet A, 17% protein) and malnourished mice (diet C, 3% protein) 3 days after infection with L. donovani. NOS2 and cNOS activity in liver and spleen tissue homogenates was determined as described in Materials and Methods. Data are expressed as the mean ± and standard error of the mean of enzyme activity per gram of tissue. Differences in mean values were determined by Student's t test. (C) Parasite burden-adjusted NOS2 activity. The splenic and hepatic NOS2 activity was adjusted to the parasite burden per milligram of the same tissue. Data are expressed as the mean and standard error of the mean (n = 11 mice per group), and significant differences between the control and malnourished mice were determined by Student's t test.