T-Cell Populations and Cytokine Expression Are Impaired in Thymus and Spleen of Protein Malnourished BALB/c Mice Infected with <i>Leishmania infantum</i>

<div><p>Visceral leishmaniasis (VL) is a parasitic infectious disease that causes significant morbidity and mortality in the tropical and subtropical regions of the world. Although infections with visceralizing <i>Leishmania</i> may be asymptomatic, factors such as undernutrition increase the likelihood of progressing to clinical disease. Protein malnutrition, the most deleterious cause of malnutrition in developing countries, has been considered as a primary risk factor for the development of clinical VL. However, data regarding the immunological basis of this association are scarce. With the aim to analyze the effects of protein malnutrition on <i>Leishmania infantum</i> infection, we used BALB/c mice subjected to control or low protein isocaloric diets. Each animal group was divided into two subgroups and one was infected with <i>L. infantum</i> resulting in four study groups: animals fed 14% protein diet (CP), animals fed 4% protein diet (LP), animals fed 14% protein diet and infected (CPi), and animals fed 4% protein diet and infected (LPi).The susceptibility to <i>L. infantum</i> infection and immune responses were assessed in terms of body and lymphoid organ weight, parasite load, lymphocyte subpopulations, and cytokine expression. LPi mice had a significant reduction of body and lymphoid organ weight and exhibited a severe decrease of lymphoid follicles in the spleen. Moreover, LPi animals showed a significant decrease in CD4⁺CD8⁺ T cells in the thymus, whereas there was an increase of CD4⁺ and CD8⁺ T cells percentages in the spleen. Notably, the cytokine mRNA levels in the thymus and spleen of protein malnourished-infected animals were altered compared to the CP mice. Protein malnutrition results in a drastic dysregulation of T cells and cytokine expression in the thymus and spleen of <i>L. infantum</i>-infected BALB/c mice, which may lead to defective regulation of the thymocyte population and an impaired splenic immune response, accelerating the events of a normal course of infection.</p></div

Protein malnutrition dysregulated cytokine expression in thymocytes and splenocytes of mice infected with <i>L. infantum</i>.

<p><i>IL-10, IL-12a, TGF-β, IL-4</i> and <i>IFNγ</i> mRNA expression levels were measured by qPCR in (<b>A</b>) thymocytes and (<b>B</b>) splenocytes of each experimental group. The values are expressed as normalized ratios between the target gene expression and the geometric median of the <i>ATP-5, GAPDH</i> and <i>CYC-1</i> genes. The values are expressed in pg/mL ± SEM. CP: animals fed 14% protein diet; LP: animals fed 4% protein diet, CPi: animals fed 14% protein diet and infected; LPi: animals fed 4% protein diet and infected. Two-way ANOVA analysis with Bonferroni pos-hoc test. Statistical differences due to diet: <b>a</b> (p<0.001), infection: <b>b</b> (p<0.05) and interaction between diet and infection: <b>c</b> (p<0.05).</p

Protein malnutrition dysregulated lymphocyte subpopulations in the thymus of mice infected with <i>L. infantum</i>.

<p>Lymphocyte subpopulations from the thymus of the experimental groups were measured using FACS analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114584#s2" target="_blank">Materials and Methods</a>. (<b>A</b>) Representative scatter plots of lymphocyte subpopulations. (<b>B</b>) Distribution of lymphocyte subsets expressed as percentage ± SEM. CP: animals fed 14% protein diet; LP: animals fed 4% protein diet, CPi: animals fed 14% protein diet and infected; LPi: animals fed 4% protein diet and infected. Two-way ANOVA analysis with Bonferroni pos-hoc test. Statistical differences due to diet: <b>a</b> (p<0.05), infection: <b>b</b> (p<0.05) and interaction between diet and infection: <b>c</b> (p<0.05).</p

Effect of protein malnutrition on tissue weight in mice infected with <i>L. infantum</i>.

<p>(<b>A</b>) Body weight gain, (<b>B</b>) thymus, (<b>C</b>) spleen and (<b>D</b>) liver weight gain at day 21 expressed as a percentage of tissue/body weight in grams ± SEM (n = 12). Two-way ANOVA analysis with Bonferroni pos-hoc test. Statistical differences due to diet: <b>a</b> (p<0.001), infection: <b>b</b> (p<0.05) and interaction between diet and infection: <b>c</b> (p<0.001). CP: animals fed 14% protein diet; LP: animals fed 4% protein diet, CPi: animals fed 14% protein diet and infected; LPi: animals fed 4% protein diet and infected.</p

Organization of the splenic white pulp in mice submitted to protein malnutrition and infected with <i>Leishmania infantum</i>.

<p>CP = animals fed 14% control protein diet; LP = animals fed 4% protein diet; CPi = animals fed 14% protein diet and infected; LPi = animals fed 4% protein diet and infected control protein and infected.</p><p>*Fisher’s exact test: CP×CPi (p = 0.60; OR: 3.5); CP×LP (p = 0.57; OR: 4.2); LP×LPi <b>(p = 0.007; OR: 25.0)</b>; CPi×LPi <b>(p = 0.001; OR: 30.0)</b>; CP×LPi <b>(p = 0.0002; OR: 105.0)</b>.</p><p>**Mann-Whitney test: CP×LP p = 0.77; CP×CPi p = 0.70; LP×LPi <b>p = 0.002</b>; CP×LPi <b>p = 0.002</b>; CPi×LPi <b>p = 0.0005</b>.</p><p>Organization of the splenic white pulp in mice submitted to protein malnutrition and infected with <i>Leishmania infantum</i>.</p

Protein malnutrition dysregulated lymphocyte subpopulations in the spleen of mice infected with <i>L. infantum</i>.

<p>Lymphocyte subpopulations from the spleen of the experimental groups were measured using FACS analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114584#s2" target="_blank">Materials and Methods</a>. (<b>A</b>) Representative scatter plots of lymphocyte subpopulations. (<b>B</b>) Distribution of lymphocyte subsets expressed as percentage ± SEM. CP: animals fed 14% protein diet; LP: animals fed 4% protein diet, CPi: animals fed 14% protein diet and infected; LPi: animals fed 4% protein diet and infected. Two-way ANOVA analysis with Bonferroni pos-hoc test. Statistical differences due to diet: <b>a</b> (p<0.001), infection: <b>b</b> (p<0.05) and interaction between diet and infection: <b>c</b> (p<0.05).</p

Positivity of infection with <i>Leishmania infantum</i> in mice submitted or not to protein malnutrition.

<p>CPi: animals fed 14% protein diet and infected; LPi: animals fed 4% protein diet and infected.</p><p>Positivity of infection with <i>Leishmania infantum</i> in mice submitted or not to protein malnutrition.</p

Histopathological alterations in the spleen and liver.

<p>Representative images of a spleen with (<b>A</b>) organized white pulp, (<b>B</b>) moderately disorganized white pulp, and (<b>C</b>) highly disorganized white pulp. The decrease or absence of lymphoid follicles is evident in B and C. (<b>D</b>) Representative image of liver from CP mice. The presence of amastigotes (arrows) in the CPi liver detected with (<b>E</b>) hematoxylin and eosin stain and (<b>F</b>) immunohistochemistry. WP: white pulp; RP: red pulp.</p

Splenic and liver parasite load in mice infected with <i>L. infantum</i>.

<p>The parasite load was determined by qPCR in liver and spleen. The number of parasites was calculated in relation to 10⁶ cells. Data represent the mean of 8–10 animals in each group. Statistical differences were analyzed by Student’s <i>t</i> test (p<0.01). CPi: animals fed 14% protein diet and infected; LPi: animals fed 4% protein diet and infected.</p

Protein malnutrition dysregulated secreted cytokine levels in the interstitial fluid of thymus and spleen of mice infected with <i>L. infantum</i>.

<p><i>IL-10</i> and <i>IL-12</i> protein levels were measured by Luminex in the interstitial fluid of (<b>A</b>) thymus and (<b>B</b>) spleen. The values are expressed in pg/mL ± SEM. CP: animals fed 14% protein diet; LP: animals fed 4% protein diet, CPi: animals fed 14% protein diet and infected; LPi: animals fed 4% protein diet and infected. Two-way ANOVA analysis with Bonferroni pos-hoc test. Statistical differences due to diet: <b>a</b> (p<0.001).</p