Deciphering the interplay between exo-erythrocytic Plasmodium berghei parasites and the host hepatic miRNA expression

Die verschiedenen Schritte während des Lebenszyklus des Malaria Parasiten bedürfen einer sehr engen Interaktion zwischen dem Parasit und dem betroffenen Gewebe des Wirts. Um das enorme Wachstum und die anschließende Differenzierung in invasive Stadien während der klinisch unauffälligen Leberstadienentwicklung gewährleisten zu können, rekrutiert der Parasit Proteine der Wirtszelle. In mehreren Studien wurden genetisch veränderte Parasiten verwendet, denen genau definierte Lebergene fehlten, um diese enge Interaktion zwischen Parasit und Wirtsleberzelle zu untersuchen. So konnte man die Abhängigkeit des Parasiten von Wirtszellfaktoren für eine erfolgreiche Leberstadienentwicklung zeigen. Außerdem waren diese genetisch veränderten Parasiten in der Lage, Immunität in dem entsprechenden Wirt zu induzieren. MicroRNAs (miRNAs) und die RNAi Maschinerie gewinnen zunehmend an Bedeutung für das Zusammenspiel von Pathogen und Wirt. Ursprünglich als Verteidigungsmechanismus des Wirts gegen Pathogene entdeckt, wurde nach und nach gezeigt, dass Pathogene in der Lage sind, die RNAi Maschinerie des Wirts für ihre eigenen Zwecke zu missbrauchen. Darüber hinaus gewinnen auch miRNAs zunehmend an Bedeutung für die Feinabstimmung sowohl bei der Auslösung von verschiedenen Erkrankungen, als auch bei ihrer Bekämpfung. Basierend auf diesen Erkenntnissen konzentrieren sich aktuelle Forschungen auf die Untersuchung des Einflusses, den eine bestehende Infektion mit Plasmodium oder Toxoplasma auf die RNAi Maschinerie und die miRNA Expression des betroffenen Wirtsgewebes hat. Das Ziel der vorliegenden Studie war das Zusammenspiel zwischen Malaria Leberstadien und der RNAi Maschinerie und miRNA Expression in der Wirtsleber im Allgemeinen zu untersuchen. Außerdem sollten die Mechanismen der durch die Immunisierung mit genetisch attenuierten (GAP) und bestrahlungsattenuierten (RAS) Parasiten hervorgerufenen Immunität analysiert werden. Dazu wurden auf der einen Seite mittels quantitativer real-time PCR (qPCR) die Transkriptionslevel von Xpo-5, Ago-2, Dicer, TRBP and Drosha in murinem Lebergewebe 24 Std. und 40 Std. nach Infektion mit PbNK65 WT, GAP und RAS Speicheldrüsen Sprozoiten bestimmt und mit den Transkriptionsleveln aus naiven Mäusen verglichen. Auf der anderen Seite wurde mit den selben Proben die hepatische Expression von miRNAs untersucht, um in ihrer Expression durch das Vorhandensein von Leberstadien der unterschiedlichen angewandten Stämme beeinflusste miRNAs der Wirtsleber zu identifizieren. Ich konnte eine Herunterregulierung der Transkription aller fünf untersuchten Komponenten feststellen, welche 40 Std. nach Infektion mit den attenuierten Parasiten besonders stark ausgeprägt war. Zusätzliche Western Blot Analysen wurden durchgeführt und bestätigten die Ergebnisse der qPCR Analyse für Xpo-5 und Drosha auch auf Proteinebene. Weiter wurden insgesamt 31 unterschiedlich regulierte hepatische Wirts miRNAs entdeckt, die für jede der verwendeten Probengruppen ein spezielles Expressionsprofil aufwiesen, welches nicht nur die einzigartigen Eigenschaften der verschiedenen eingesetzten Stämme widerspiegelte, sondern auch die zuvor beobachteten Veränderungen in den Transkriptionsraten der RNAi Maschinerie Komponenten. Zwei interessante miRNAs, miR-21 und miR-155, wurden hinsichtlich einer möglichen biologischen Funktion genauer untersucht. Nachdem durch ex vivo Bildgebung gezeigt werden konnte, dass beide miRNAs tatsächlich in infiziertem Lebergewebe überexpremiert werden, wurde ihre Beteiligung am Hervorrufen von Immunantworten durch Immunisierung mit GAP und RAS untersucht. Dazu wurden beide miRNAs unabhängig von einander in vivo in der Mausleber herunterreguliert und dann das Potenzial von GAP und RAS Immunisierungen beobachtet. Diese Experimente zeigten, dass miR-21 und miR-155 an der durch GAP Immunisierung hervorgerufenen Immunität beteiligt sind

The aryl hydrocarbon receptor in T cells contributes to sustaining oral tolerance against ovalbumin in a mouse model

Oral tolerance (OT) towards antigens encountered in the gut is a vital immune function of gut immunity. Experimental models can demonstrate OT efficacy by feeding of a protein followed by peripheral immunization and measuring the specific antibody titer. We had previously shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a xenobiotic high-affinity aryl hydrocarbon receptor (AhR)-ligand, destabilized OT against ovalbumin (OVA) in mice. AhR is involved in the development, differentiation and function of immune cells, and highly expressed in gut epithelial cells and gut immune cells. We here used AhR-deficient mice to study the role of AhR in OT further. We show that complete AhR-deficiency undermines the stability of oral tolerance against OVA upon multiple immunizations, despite no renewed oral encounter with the antigen. This OT destabilization is accompanied by significant changes in IL10 and TGFß RNA in the gut tissue. Using conditional AhR-deficient mouse lines, we identify T cells as the major responsible immune cell type in this context. Our findings add to knowledge that lack of AhR signaling in the gut impairs important gut immune functions

Impaired glucose and lipid metabolism in ageing aryl hydrocarbon receptor deficient mice

Disturbed homeostasis of glucose and lipid metabolism are dominant features of the so-called metabolic syndrome (MetS) and can increase the risk for the development of type 2 diabetes (T2D), a severe metabolic disease. T2D prevalence increases with age. The aryl hydrocarbon receptor (AHR) is a sensor of small molecules including dietary components. AHR has been identified as potential regulator of glucose homeostasis and lipid metabolism. Epidemiologically, exposure to xenobiotic AHR ligands such as polycyclic aromatic hydrocarbons is linked to T2D. We assess here the potential role of the AHR in disturbances of glucose and lipid metabolism in young (age 2-5 months) and old (age > 1,5 years) AHR-deficient (AHR KO) mice. Fasted young wildtype (WT) and AHR-KO mice displayed similar blood glucose kinetics after challenge with intra-peritoneal glucose injection. However, old AHR-KO mice showed lower tolerance than WT to i.p. administered glucose, i.e. glucose levels rose higher and returned more slowly to normal levels. Old mice had overall higher insulin levels than young mice, and old AHR-KO had a somewhat disturbed insulin kinetic in the serum after glucose challenge. Surprisingly, young AHR-KO mice had significantly lower triglycerides, cholesterol, high density lipoprotein values than WT, i.e., a dyslipidemic profile. With ageing, AHR-KO and WT mice did not differ in these lipid levels, except for slightly reduced levels of triglycerides and cholesterol. In conclusion, our findings in AHR KO mice suggest that AHR expression is relevant for the maintenance of glucose and lipid homeostasis in old mice

Impaired Glucose and Lipid Metabolism in Ageing Aryl Hydrocarbon Receptor Deficient Mice

Disturbed homeostasis of glucose and lipid metabolism are dominant features of the so-called metabolic syndrome (MetS) and can increase the risk for the development of type 2 diabetes (T2D), a severe metabolic disease. T2D prevalence increases with age. The aryl hydrocarbon receptor (AHR) is a sensor of small molecules including dietary components. AHR has been identified as potential regulator of glucose homeostasis and lipid metabolism. Epidemiologically, exposure to xenobiotic AHR ligands such as polycyclic aromatic hydrocarbons is linked to T2D. We assess here the potential role of the AHR in disturbances of glucose and lipid metabolism in young (age 2-5 months) and old (age > 1,5 years) AHR-deficient (AHR KO) mice. Fasted young wildtype (WT) and AHR-KO mice displayed similar blood glucose kinetics after challenge with intra-peritoneal glucose injection. However, old AHR-KO mice showed lower tolerance than WT to i.p. administered glucose, i.e. glucose levels rose higher and returned more slowly to normal levels. Old mice had overall higher insulin levels than young mice, and old AHR-KO had a somewhat disturbed insulin kinetic in the serum after glucose challenge. Surprisingly, young AHR-KO mice had significantly lower triglycerides, cholesterol, high density lipoprotein values than WT, i.e., a dyslipidemic profile. With ageing, AHR-KO and WT mice did not differ in these lipid levels, except for slightly reduced levels of triglycerides and cholesterol. In conclusion, our findings in AHR KO mice suggest that AHR expression is relevant for the maintenance of glucose and lipid homeostasis in old mice

In vitro and in vivo silencing of plasmodial dhs and eIf-5a genes in a putative, non-canonical RNAi-related pathway

Abstract Background Deoxyhypusine synthase (DHS) catalyzes the first step in hypusine biosynthesis of eukaryotic initiation factor 5A (eIF-5A) in Plasmodium falciparum. Target evaluation of parasitic DHS has recently been performed with CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease. CNI-1493 prevented infected mice from experimental cerebral malaria by decreasing the levels in hypusinated eIF-5A and serum TNF, implicating a link between cytokine signaling and the hypusine pathway. Therefore we addressed the question whether either DHS itself or eIF-5A is required for the outcome of severe malaria. In a first set of experiments we performed an in vitro knockdown of the plasmodial eIF-5A and DHS proteins by RNA interference (RNAi) in 293 T cells. Secondly, transfection of siRNA constructs into murine Plasmodium schizonts was performed which, in turn, were used for infection. Results 293 T cells treated with plasmodial DHS- and eIF-5A specific siRNAs or control siRNAs were analyzed by RT-PCR to determine endogenous dhs -and eIF-5A mRNA levels. The expressed DHS-shRNA and EIF-5A-shRNA clearly downregulated the corresponding transcript in these cells. Interestingly, mice infected with transgenic schizonts expressing either the eIF-5A or dhs shRNA showed an elevated parasitemia within the first two days post infection which then decreased intermittently. These results were obtained without drug selection. Blood samples, which were taken from the infected mice at day 5 post infection with either the expressed EIF-5A-shRNA or the DHS-shRNA were analyzed by RT-PCR and Western blot techniques, demonstrating the absence of either the hypusinated form of eIF-5A or DHS. Conclusions Infection of NMRI mice with schizonts from the lethal P. berghei ANKA wildtype strain transgenic for plasmodial eIF-5A-specific shRNA or DHS-specific shRNA resulted in low parasitemia 2–9 days post infection before animals succumbed to hyperparasitemia similar to infections with the related but non-lethal phenotype P. berghei strain NK65. RT-PCR and Western blot experiments performed with blood from the transfected erythrocytic stages showed that both genes are important for the proliferation of the parasite. Moreover, these experiments clearly demonstrate that the hypusine pathway in Plasmodium is linked to human iNos induction.</p

Indole-3-carbinol, a plant nutrient and AhR-Ligand precursor, supports oral tolerance against OVA and improves peanut allergy symptoms in mice

In general, dietary antigens are tolerated by the gut associated immune system. Impairment of this so-called oral tolerance is a serious health risk. We have previously shown that activation of the ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) by the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects both oral tolerance and food allergy. In this study, we determine whether a common plant-derived, dietary AhR-ligand modulates oral tolerance as well. We therefore fed mice with indole-3-carbinole (I3C), an AhR ligand that is abundant in cruciferous plants. We show that several I3C metabolites were detectable in the serum after feeding, including the high-affinity ligand 3,3´-diin-dolylmethane (DIM). I3C feeding robustly induced the AhR-target gene CYP4501A1 in the intestine; I3C feeding also induced the aldh1 gene, whose product catalyzes the formation of retinoic acid (RA), an inducer of regulatory T cells. We then measured parameters indicating oral tolerance and severity of peanut-induced food allergy. In contrast to the tolerance-breaking effect of TCDD, feeding mice with chow containing 2 g/kg I3C lowered the serum anti-ovalbumin IgG1 response in an experimental oral tolerance protocol. Moreover, I3C feeding attenuated symptoms of peanut allergy. In conclusion, the dietary compound I3C can positively influence a vital immune function of the gut

Protective efficacy and safety of liver stage attenuated malaria parasites

During the clinically silent liver stage of a Plasmodium infection the parasite replicates from a single sporozoite into thousands of merozoites. Infection of humans and rodents with large numbers of sporozoites that arrest their development within the liver can cause sterile protection from subsequent infections. Disruption of genes essential for liver stage development of rodent malaria parasites has yielded a number of attenuated parasite strains. A key question to this end is how increased attenuation relates to vaccine efficacy. Here, we generated rodent malaria parasite lines that arrest during liver stage development and probed the impact of multiple gene deletions on attenuation and protective efficacy. In contrast to P. berghei strain ANKA LISP2(-) or uis3(-) single knockout parasites, which occasionally caused breakthrough infections, the double mutant lacking both genes was completely attenuated even when high numbers of sporozoites were administered. However, different vaccination protocols showed that LISP2(-) parasites protected better than uis3(-) and double mutants. Hence, deletion of several genes can yield increased safety but might come at the cost of protective efficacy

Quantification of I3C and metabolites in mouse serum after oral uptake.

<p>Mice were fed for up to 7 days with chow containing 2 g/kg I3C. For the 15 minute value, mice received 5 mg/I3C per mouse diluted in 10%DMSO/olive oil by gavage. Serum samples were prepared and analyzed for the presence of I3C, DIM, and ICZ. Sera were spiked with 4-methoxyindole as the internal standard to calibrate for possible differences in extraction efficiency. Note that I3C is detectable only after the bolus feeding, but not when I3C was given in the chow, and is thus eaten in smaller amounts over the entire day. Y-axis: arbitrary units.</p

I3C in the diet licenses oral tolerance.

<p>Mice were fed a placebo (black bars) or I3C containing diet (white bars) and were either not tolerized (PBS = control) or tolerized with OVA (dotted bars). Mice were immunized and then boosted 2 (A), 3 (B), and 4 weeks (C) later. Antibody titers were determined at the day of each boost and one week after the third boost (D). Antibody titers are depicted as % of placebo/PBS-treated animals that were set to 100%. Data are pooled from three independent experiments. N = 12–13 mice/group. Mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test. *≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.</p

CYP induction in small intestine.

<p>A-D: Mice were fed by gavage with TCDD, I3C or DMSO (as solvent for TCDD) at the indicated doses. Shown is <i>cyp1a1</i> expression relative to the housekeeping gene <i>rps6</i> in the three sections of the small intestine 4 hours after gavage (A, B), or 24 hours after gavage (C, D). Note the scale differences for TCDD and I3C. N = 3; Mean ± SEM. Two-way ANOVA followed by Sidak’s multiple comparison test. *≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001. E: Mice were fed for 49 days with chow containing 2 g/kg I3C. On day 49, RNA was isolated from small intestine and <i>cyp1a1</i> expression was analyzed by quantitative PCR. The results of two pooled independent experiments are shown. N = 11–15. Mean ± SEM. Two-way ANOVA followed by Sidak’s multiple comparison test. *≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.</p