Emerging Role of HMGB1 in the Pathogenesis of Schistosomiasis Liver Fibrosis

In chronic schistosomiasis, liver fibrosis is linked to portal hypertension, which is a condition associated with high mortality and morbidity. High mobility group box 1 (HMGB1) was originally described as a nuclear protein that functions as a structural co-factor in transcriptional regulation. However, HMGB1 can also be secreted into the extracellular milieu under appropriate signal stimulation. Extracellular HMGB1 acts as a multifunctional cytokine that contributes to infection, injury, inflammation, and immune responses by binding to specific cell-surface receptors. HMGB1 is involved in fibrotic diseases. From a clinical perspective, HMGB1 inhibition may represent a promising therapeutic approach for treating tissue fibrosis. In this study, we demonstrate elevated levels of HMGB1 in the sera in experimental mice or in patients with schistosomiasis. Using immunohistochemistry, we demonstrated that HMGB1 trafficking in the hepatocytes of mice suffering from acute schistosomiasis was inhibited by Glycyrrhizin, a well-known HMGB1 direct inhibitor, as well as by DIC, a novel and potential anti-HMGB1 compound. HMGB1 inhibition led to significant downregulation of IL-6, IL4, IL-5, IL-13, IL-17A, which are involved in the exacerbation of the immune response and liver fibrogenesis. Importantly, infected mice that were treated with DIC or GZR to inhibit HMGB1 pro-inflammatory activity showed a significant increase in survival and a reduction of over 50% in the area of liver fibrosis. Taken together, our findings indicate that HMGB1 is a key mediator of schistosomotic granuloma formation and liver fibrosis and may represent an outstanding target for the treatment of schistosomiasis

Purinergic P2Y12 Receptor Activation in Eosinophils and the Schistosomal Host Response

Identifying new target molecules through which eosinophils secrete their stored proteins may reveal new therapeutic approaches for the control of eosinophilic disorders such as host immune responses to parasites. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in this cell type and its involvement in eosinophilic inflammation remain unknown. Here, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by Schistosoma mansoni (S. mansoni) infection. We found that adenosine 5’-diphosphate (ADP) induced human eosinophils to secrete eosinophil peroxidase (EPO) in a P2Y12R dependent manner. However, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro. In vivo, C57Bl/6 mice were infected with cercariae of the Belo Horizonte strain of S. mansoni. Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the granulomatous hepatic area and the eosinophilic infiltrate, collagen deposition and IL-13/IL-4 production in the liver without affecting the parasite oviposition. As found for humans, murine eosinophils also express the P2Y12R. P2Y12R inhibition increased blood eosinophilia, whereas it decreased the bone marrow eosinophil count. Our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection

Resveratrol Reverses Functional Chagas Heart Disease in Mice

<div><p>Chronic chagasic cardiomyopathy (CCC) develops years after acute infection by <i>Trypanosoma cruzi</i> and does not improve after trypanocidal therapy, despite reduction of parasite burden. During disease, the heart undergoes oxidative stress, a potential causative factor for arrhythmias and contractile dysfunction. Here we tested whether antioxidants/ cardioprotective drugs could improve cardiac function in established Chagas heart disease. We chose a model that resembles B1-B2 stage of human CCC, treated mice with resveratrol and performed electrocardiography and echocardiography studies. Resveratrol reduced the prolonged PR and QTc intervals, increased heart rates and reversed sinus arrhythmia, atrial and atrioventricular conduction disorders; restored a normal left ventricular ejection fraction, improved stroke volume and cardiac output. Resveratrol activated the AMPK-pathway and reduced both ROS production and heart parasite burden, without interfering with vascularization or myocarditis intensity. Resveratrol was even capable of improving heart function of infected mice when treatment was started late after infection, while trypanocidal drug benznidazole failed. We attempted to mimic resveratrol’s actions using metformin (AMPK-activator) or tempol (SOD-mimetic). Metformin and tempol mimicked the beneficial effects of resveratrol on heart function and decreased lipid peroxidation, but did not alter parasite burden. These results indicate that AMPK activation and ROS neutralization are key strategies to induce tolerance to Chagas heart disease. Despite all tissue damage observed in established Chagas heart disease, we found that a physiological dysfunction can still be reversed by treatment with resveratrol, metformin and tempol, resulting in improved heart function and representing a starting point to develop innovative therapies in CCC.</p></div

Effects of metformin and tempol on heart function, lipid peroxidation and parasite burden of chronically infected mice.

<p>Infected mice were treated with peroral metformin (Met), tempol (Tmp), or oral vehicle (VEH) from 60–90 dpi. Infected mice treated i.p. with resveratrol (RSV) or vehicle (VEH) were kept as controls, as well as non-infected mice. Heart function, oxidative damage and parasite burden were assessed at 90 dpi. (A) heart rate, PR interval, P duration, QRS duration, QTc interval. Mice per group: NI (n = 11), VEH (n = 7), Met (n = 9), Tmp (n = 9). Met are representative of 2 similar experiments. (B) Pre- and post-treatment ECG tracings. Picture illustrate the reversion of abnormalities in Met and Tmp, but not in VEH group, in three individual mice from each group (#1–9). (C) Left ventricle ejection fraction (LVEF), stroke volume, right ventricle area (RV area); left ventricle area (LV area). Mice per group: NI (n = 10), VEH (n = 8), Met (n = 9), Tmp (n = 8). (D), Lipid peroxidation (TBARs) in heart extracts. Mice per group: NI (n = 3), VEH (n = 3), Met (n = 7–9), Tmp (n = 8).(E) Relative amount of parasites (TcS18) per host DNA (GADPH) in individual heart tissue. Mice per group: NI (n = 6), VEH (n = 5), Met (n = 6), Tmp (n = 5). *, different from NI; §, different from VEH, #, different from oral VEH. P range: * § #, P≤0.05, ** §§ ##, P<0.01, *** §§§ ###, P<0.005</p

Resveratrol reverses heart-pumping dysfunction in mice chronically infected with <i>T</i>. <i>cruzi</i>.

<p>(A) <i>Upper panel</i>: echocardiography comparing heart function of non-infected (NI) and infected (INF) mice at 60 dpi (before treatment). Left ventricle ejection fraction (LVEF), left ventricle stroke volume and cardiac output. NI (n = 4), INF (n = 14), pooled from two independent experiments. <i>Lower panel</i>: at 90 dpi (after treatment): similar heart function parameters are shown for infected mice treated with vehicle (VEH, n = 20), resveratrol (RSV, n = 20) or for non-infected mice (NI, n = 23), pooled from three independent experiments. (B) Similar parameters shown for mice treated from 120 to 160 dpi, at 170 dpi. NI (n = 5), VEH (n = 5), RSV (n = 7), Bzd (n = 6). *, different from NI; §, different from VEH, ¥, different from resveratrol. P range: * § ¥, P≤0.05, ** §§, P<0.01, *** §§§, P<0.005</p

Diagrammatic illustration of the effects of resveratrol on established Chagas heart disease.

<p>Treatment with resveratrol, an AMPK activator (metformin), or a ROS scavenger (tempol) were capable of ameliorating cardiac function in Chagas disease. The drugs reduced the incidence of sinus arrhythmia (SA), atrial abnormalities, intra-atrial and interatrial block (AIb), sinoatrial block (SAb), second-degree atrioventricular block (AVb2), reduced the QT and PR intervals, increased the heart rate, increased ejection fraction (EF), stroke volume (SV), and cardiac output (CO). The 3D ECG tracing was chosen from a mouse before and after treatment with resveratrol.</p

Resveratrol reverses ECG abnormalities in mice chronically infected with <i>T</i>. <i>cruzi</i>.

<p>Mice were individually identified, infected with Colombian-strain and treated with either resveratrol (RSV) or vehicle (VEH) from 60–90 dpi. Non-infected (NI) mice were kept as controls. (A), Representative tracings for each group at 90 dpi. (B-F), Left: evolution of ECG intervals for each individual mouse during the 60–90 dpi period. P-range is shown for paired comparisons. The gray shade represents the range of values for NI. Right: the ECG intervals at 90 dpi. P-range is shown for the unpaired comparisons. (G)<b>,</b> Representative ECG tracings for 3 individual mice per group before and after treatment. Note that atrial and atrioventricular conduction disorders (intra-atrial/ interatrial block, AIb, #1,#4), sinoatrial block (SAb, #2,#5), and second-degree atrioventricular block (AVb2, #3,#6), subsided in response to resveratrol, but not in response to vehicle. (H), incidence of sinus arrhythmia (SA, left) and atrial/ atrioventricular conduction disorders (right): sinoatrial block (SAb), intra-atrial/ interatrial block (IAb), and second-degree atrioventricular block (AVb2). Mice were pooled from six independent experiments (NI n = 46; VEH n = 49; RSV n = 47). Error bars indicate mean±SEM. *, different from NI; §, different from VEH. P range: *, § P≤0.05, ** §§, P<0.01, *** §§§ ¥¥¥, P<0.005</p

P2Y12R blockade does not alter the course of <i>S</i>. <i>mansoni</i> parasite oviposition.

<p>Mice were infected with 60 <i>S</i>. <i>mansoni</i> cercariae through a percutaneous route, and the livers and intestines were collected 55 days post-infection. Graphs show the number of eggs per gram tissue within (A) the liver and (B) intestines. Data represents the mean ± SE; 5–8 infected mice were used as individual tissue donors and represents animals from at least 2 independent infection studies.</p

P2Y12R blockade impairs hepatic granulomatous eosinophilia.

<p>Representative granuloma images from infected mice (Ai) untreated or (Aii) treated with clopidogrel. Slide sections were stained with Syrius red (modified method) and examined under a light microscope. (B) The number of eosinophils per granuloma area were calculated from images of 10–20 granulomas acquired for each individual mouse. Data represent the mean ± SE and were analyzed by Student’s <i>t</i> test. (*p<0.05). <i>n</i> = 10 mice/group and represents animals from at least 2 independent infection studies. (C) Immunofluorescence showing Siglec-F and P2Y12R staining of cytospin smears of murine eosinophil-enriched suspensions. P2Y12R, green; Siglec-F, red; Ab = antibody. Data are from one experiment, representative of 3 experiments. (D) Flow cytometry dot plots showing isotype control (left panel) and Siglec-F versus P2Y12R staining (right panel) of mice bone marrow derived eosinophils. Data are from one experiment, representative of 3 experiments.</p