Dietary Habits and Intestinal Immunity: From Food Intake to CD4+ TH Cells

Dietary habits have a profound impact on intestinal homeostasis and in general on human health. In Western countries, high intake of calories derived from fried products, butter and processed meat is favored over dietary regimens rich in fruits and vegetables. This type of diet is usually referred to as Western-type diet (WTD) and it has been associated with several metabolic and chronic inflammatory conditions of the gastrointestinal tract. In this review, we describe how WTD promotes intestinal and extra-intestinal inflammation and alters mucosal immunity acting on CD4+ T cells in a microbiota-dependent or –independent fashion, ultimately leading to higher susceptibility to infectious and autoimmune diseases. Moreover, summarizing recent findings, we propose how dietary supplementation with fiber and vitamins could be used as a tool to modulate CD4+ T cell phenotype and function, ameliorating inflammation and restoring mucosal homeostasis

A Gas Chromatography Mass Spectrometry-Based Method for the Quantification of Short Chain Fatty Acids

Short Chain Fatty Acids (SCFAs) are produced by the gut microbiota and are present in varying concentrations in the intestinal lumen, in feces but also in the circulatory system. By interacting with different cell types in the body, they have a great impact on host metabolism and their exact quantification is indispensable. Here, we present a derivatization-free method for the gas chromatography mass spectrometry (GC-MS) based quantification of SCFAs in plasma, feces, cecum, liver and adipose tissue. SCFAs were extracted using ethanol and concentrated by alkaline vacuum centrifugation. To allow volatility for separation by GC, samples were acidified with succinic acid. Analytes were detected in selected ion monitoring (SIM) mode and quantified using deuterated internal standards and external calibration curves. Method validation rendered excellent linearity (R2 > 0.99 for most analytes), good recovery rates (95–117%), and good reproducibility (RSD: 1–4.5%). Matrix effects were ruled out in plasma, feces, cecum, liver and fat tissues where most abundant SCFAs were detected and accurately quantified. Finally, applicability of the method was assessed using samples derived from conventionally raised versus germ-free mice or mice treated with antibiotics. Altogether, a reliable, fast, derivatization-free GC-MS method for the quantification of SCFAs in different biological matrices was developed allowing for the study of the (patho)physiological role of SCFAs in metabolic health

The P2X7 ion channel is dispensable for energy and metabolic homeostasis of white and brown adipose tissues

Several studies suggest a role of extracellular adenine nucleotides in regulating adipose tissue functions via the purinergic signaling network. Metabolic studies in mice with global deletion of the purinergic receptor P2X7 on the C57BL/6 background indicate that this receptor has only a minor role in adipose tissue for diet-induced inflammation or cold-triggered thermogenesis. However, recent data show that a polymorphism (P451L) present in C57BL/6 mice attenuates P2X7 receptor function, whereas BALB/c mice express the fully functional P451 allele. To determine the potential role of P2rx7 under metabolic and thermogenic stress conditions, we performed comparative studies using male P2rx7 knockout (KO) and respective wild-type controls on both BALB/c and C57BL/6 backgrounds. Our data show that adipose P2rx7 mRNA levels are increased in obese mice. Moreover, P2rx7 deficiency results in reduced levels of circulating CCL2 and IL6 with a moderate effect on gene expression of pro-inflammatory markers in white adipose tissue and liver of BALB/c and C57BL/6 mice. However, P2X7 expression does not alter body weight, insulin resistance, and hyperglycemia associated with high-fat diet feeding on both genetic backgrounds. Furthermore, deficiency of P2rx7 is dispensable for energy expenditure at thermoneutral and acute cold exposure conditions. In summary, these data show that-apart from a moderate effect on inflammatory cytokines-P2X7 plays only a minor role in inflammatory and thermogenic effects of white and brown adipose tissue even on the BALB/c background

Abscisic acid stimulates the release of insulin and of GLP-1 in the rat perfused pancreas and intestine

Aims: Previous results indicate that nanomolar concentrations of abscisic acid (ABA) stimulate insulin release from \u3b2-pancreatic cells in vitro and that oral ABA at 50 mg/kg increases plasma GLP-1 in the fasted rat. The aim of this study was to test the effect of ABA on the perfused rat pancreas and intestine, to verify the insulin- and incretin-releasing actions of ABA in controlled physiological models. Materials and methods: Rat pancreas and small intestine were perfused with solutions containing ABA at high-micromolar concentrations, or control secretagogues. Insulin and GLP-1 concentrations in the venous effluent were analysed by radioimmunoassay, and ABA levels were determined by ELISA. Results: High micromolar concentrations of ABA induced GLP-1 secretion from the proximal half of the small intestine and insulin secretion from pancreas. GLP-1 stimulated ABA secretion from pancreas in a biphasic manner. Notably, a positive correlation was found between the ABA area under the curve (AUC) and the insulin AUC upon GLP-1 administration. Conclusion: Our results indicate the existence of a cross talk between GLP-1 and ABA, whereby ABA stimulates GLP-1 secretion, and vice versa. Release of ABA could be considered as a new promising molecule in the strategy of type 2 diabetes treatment and as a new endogenous hormone in the regulation of glycaemia

Increased systemic replication of <i>S.</i> Typhimurium (<i>S</i>Tm) during concurrent <i>P. yoelii</i> (<i>Py</i>) infection.

<p><b>A,</b> Parasitemia and anemia in <i>P. yoelii</i>-infected mice (n = 4). Arrows indicate time points at which <i>P. yoelii</i> and <i>S.</i> Typhimurium were inoculated. <b>B,</b> Comparison of parasitemia at 14 d after <i>P. yoelii</i> infection in <i>P. yoelii</i>-infected mice and mice co-infected for 4 d with <i>S.</i> Typhimurium (n = 5). Data are shown as mean ± SEM. <b>C,</b> Colonization (CFU) of the liver at 2 and 4 days after IG infection of <i>P. yoelii</i>-infected or uninfected CBA mice with <i>S.</i> Typhimurium (n = 5–10). Results are from 2 independent experiments. <b>D,</b> CFU of <i>S.</i> Typhimurium in the blood 4 days after IG infection of CBA mice (n = 5). Panels, B, C and D are compiled from the two independent experiments. <b>E–F,</b> CFU in liver (<b>E</b>) and blood (<b>F</b>) at 2 and 3 days after IP infection of <i>P. yoelii</i>-infected or uninfected CBA mice (n = 5). Dots represent individual mice and bars represent the mean ± SEM. Statistical significance was determined using an unpaired Student's <i>t</i> test on log-transformed values and is indicated as *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Liver CD11b⁺ macrophages exhibit increased association with <i>S.</i> Typhimurium and an altered phenotype during malaria parasite co-infection.

<p><b>A,</b> CD11b⁺ liver cells were enriched by positive selection from <i>S.</i> Typhimurium-infected and co-infected CBA mice at 4 days post-<i>S.</i> Typhimurium infection (n = 4). CFU of <i>S.</i> Typhimurium was enumerated from 10⁶ CD11b⁺ liver cells. CD11b⁺ liver cells, <b>B,</b> Expression of <i>Il10</i>, <i>Ym1</i>, <i>Fizz1</i>, and <i>Arg1</i> in CD11b⁺ liver cells was determined by qRT-PCR. Data are expressed as fold change over mock-infected control. All mice were co-infected as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004049#ppat-1004049-g001" target="_blank">Fig. 1A</a>. Dots represent individual mice and bars represent the mean ± SEM. Statistical significance was determined using an unpaired Student's <i>t</i> test (<b>A</b>) or one-way ANOVA with Tukey's post test (<b>B</b>) on log-transformed values and is indicated as *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Macrophage/neutrophil derived IL-10 contributes to increased systemic <i>S.</i> Typhimurium burden during concurrent malaria parasite infection.

<p>Mice conditionally deficient for IL-10 expression on myeloid cells (<i>Il10^f/f LysM-cre</i>) or Cre-negative littermate controls were infected with <i>S.</i> Typhimurium or co-infected with <i>S.</i> Typhimurium and <i>P. yoelii</i>. Bacterial colonization was assessed 2 days after IP inoculation with <i>S.</i> Typhimurium and 12 days after <i>P. yoelii</i> inoculation (n = 5–6). <b>A,</b> Colonization of liver tissue (left panel) and blood (right panel) by <i>S.</i> Typhimurium. <b>B,</b> Parasitemia in Cre^+/− and Cre^−/− mice co-infected with <i>P. yoelii</i> and <i>S.</i> Typhimurium <b>C,</b>. Expression of <i>Il10</i> in livers of <i>Il10^f/f LysM-cre</i> mice or littermate controls at 12 after <i>P. yoelii</i> infection and 2 days after <i>S.</i> Typhimurium infection. Bars represent the mean +SEM (n = 5). Dots represent individual mice and bars represent the mean ± SEM. Data are compiled from 2 independent experiments. Statistical significance was determined using an unpaired Student's <i>t</i> test on log-transformed values and is indicated as *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Malaria parasite-induced IL-10 acts on myeloid cells.

<p>Mice conditionally deficient for IL-10 receptor (Il10R) on myeloid cells (<i>Il10R^f/f LysM-cre</i>) or Cre-negative littermate controls were infected with <i>S.</i> Typhimurium or co-infected with <i>S.</i> Typhimurium and <i>P. yoelii</i>. Bacterial colonization was assessed 2 days after IP inoculation with <i>S.</i> Typhimurium and 12 days after <i>P. yoelii</i> inoculation (n = 5–8). <b>A,</b> Colonization of liver tissue by <i>S.</i> Typhimurium. <b>B, </b><i>S.</i> Typhimurium bacteremia. <b>C,</b> Parasitemia in Cre^+/− and Cre^−/− mice co-infected with <i>P. yoelii</i> and <i>S.</i> Typhimurium. Dots represent individual mice and bars represent the mean ± SEM. Data are compiled from 2 independent experiments. Statistical significance was determined using an unpaired Student's <i>t</i> test on log-transformed values and is indicated as *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Decreased neutrophil-mediated pathology in the liver of co-infected mice.

<p><b>A,</b> Quantification of inflammatory lesions in the liver and representative micrographs of H&E stained liver tissue from mock-infected mice, mice infected with <i>P. yoelii</i>, or co-infected mice 4 days after IG inoculation of <i>S.</i> Typhimurium in CBA mice (n = 5–10). Arrows represent pyogranulomatous lesions and arrowheads represent perivascular monocytic infiltrates. Results are from the experiment shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004049#ppat-1004049-g001" target="_blank">figure 1B</a>. <b>B,</b> Representative flow cytometry plots for neutrophil frequency in singlet live CD3⁻ B220⁻ NK1.1⁻ CD11b⁺ liver cells. Right panel shows quantification of neutrophils from <i>S.</i> Typhimurium and co-infected mice (n = 4–5). Dotted line represents mock-infected mice (n = 2). The gating strategy used for generation of these results is shown in <b>Fig. S2 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004049#ppat.1004049.s001" target="_blank">Text S1</a></b>. <b>C,</b> Number of neutrophils (singlet live CD3⁻ B220⁻ NK1.1⁻ CD11b⁺ Ly6G⁺) per 4×10⁶ liver cells determined by AccuCount beads. Results are from data shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004049#ppat-1004049-g002" target="_blank">figure 2D</a>. <b>D,</b> Number of circulating neutrophils (K/µl) determined by complete blood counts. Results are from data shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004049#ppat-1004049-g002" target="_blank">figure 2C</a>. <b>E,</b> Expression of <i>Cxcl1</i>, <i>Lcn2</i> and <i>Il10</i> in liver tissue of CBA mice 4 days after IG inoculation with <i>S.</i> Typhimurium (n = 4–9). Data expressed as fold change over mock-infected control. Results are from the experiment shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004049#ppat-1004049-g001" target="_blank">figure 1B</a>. <b>F,</b> Circulating IL-10 measured 2 days after IG infection with <i>S.</i> Typhimurium. Results are from the experiment shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004049#ppat-1004049-g001" target="_blank">figure 1B</a>. Data bars represent the mean +SEM. Statistical significance was determined using an unpaired Student's <i>t</i> test (<b>A–D, F</b>) or ANOVA with Tukey's post test (<b>E</b>) on log-transformed values and is indicated as *, <i>P</i><0.05; **, <i>P</i><0.01.</p

IL-10 contributes to increased systemic loads of <i>S.</i> Typhimurium in malaria parasite-infected CBA mice.

<p><b>A, </b><i>S.</i> Typhimurium and co-infected CBA mice as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004049#ppat-1004049-g001" target="_blank">Fig. 1A</a>, were depleted of IL-10 using a blocking antibody, or treated with a control antibody. At 2 d post-<i>S.</i> Typhimurium infection, CFU of <i>S.</i> Typhimurium were enumerated in liver and blood (n = 4–5). Statistical significance was determined using an unpaired Student's <i>t</i> test on log-transformed values, or a Mann-Whitney U test for groups that include all zero values, and is indicated as *, <i>P</i><0.05; **, <i>P</i><0.01. <b>B,</b> Mice were treated with either an anti-RBC antibody to induce anemia, recombinant IL-10 (rIL-10) or both. At 4 days post-<i>S.</i> Typhimurium infection, CFU were enumerated in liver and blood (n = 5). Dots represent individual mice and bars represent the mean ± SEM. Brackets indicate the contributions of anemia and IL-10 to increased bacterial colonization. <b>C,</b> Levels of anemia in experimental groups from (<b>B</b>) at 4 d post IG infection. The number of circulating RBC in mice infected with <i>P. yoelii</i> at 14 d after inoculation is given as a reference. Statistical significance was determined using one-way ANOVA with a Tukey's post test *, <i>P</i><0.05; **, <i>P</i><0.01.</p