Nicolás Eric Ponce

Toll-like receptor-2 and interleukin-6 mediate cardiomyocyte protection from apoptosis during Trypanosoma cruzi murine infection

Local innate immunity plays a key role in initiating and coordinating homeostatic and defense responses in the heart. We have previously reported that the cardiotropic parasite Trypanosoma cruzi, the etiological agent of Chagas disease, protects cardiomyocytes against growth factor deprivation-induced apoptosis. In this study, we investigated cardiomyocyte innate immune response to T. cruzi infection and its role in cellular protection from apoptosis. We found that Toll-like receptor (TLR) 2-expressing cells were strongly increased by the parasite in BALB/c neonatal mouse cardiomyocyte cultures. Using a dominant-negative system, we showed that TLR2 mediated cardiomyocyte survival and the secretion of interleukin (IL) 6, which acted as an essential anti-apoptotic factor. Moreover, IL6 released by infected cells, as well as the recombinant bioactive cytokine, induced the phosphorylation of the signal transducers and activators of transcription-3 (STAT3) in cultured cardiomyocytes. In accord with the in vitro results, during the acute phase of the infection, TLR2 expression increased 2.9-fold and the anti-apoptotic factor Bcl-2 increased 4.5-fold in the cardiac tissue. We have clearly shown a cross-talk between the intrinsic innate response of cardiomyocytes and the pro-survival effect evoked by the parasite.

IL-6 promotes M2 macrophage polarization by modulating purinergic signaling and regulates the lethal release of nitric oxide during Trypanosoma cruzi infection

The production of nitric oxide (NO) is a key defense mechanism against intracellular pathogens but it must be tightly controlled in order to avoid excessive detrimental oxidative stress. In this study we described a novel mechanism through which interleukin (IL)-6 mediates the regulation of NO release induced in response to Trypanosoma cruzi infection. Using a murine model of Chagas disease, we found that, in contrast to C57BL/6 wild type (WT) mice, IL-6-deficient (IL6KO) mice exhibited a dramatic increase in plasma NO levels concomitant with a significantly higher amount of circulating IL-1β and inflammatory monocytes. Studies on mouse macrophages and human monocytes, revealed that IL-6 decreased LPS-induced NO production but this effect was abrogated in the presence of anti-IL-1β and in macrophages deficient in the NLRP3 inflammasome. In accordance, while infected WT myocardium exhibited an early shift from microbicidal/M1 to anti-inflammatory/M2 macrophage phenotype, IL6KO cardiac tissue never displayed a dominant M2 macrophage profile that correlated with decreased expression of ATP metabolic machinery and a lower cardiac parasite burden. The deleterious effects of high NO production-induced oxidative stress were evidenced by enhanced cardiac malondialdehyde levels, myocardial cell death and mortality. The survival rate was improved by the treatment of IL-6-deficient mice with a NO production-specific inhibitor. Our data revealed that IL-6 regulates the excessive release of NO through IL-1β inhibition and determines the establishment of an M2 macrophage profile within infected heart tissue.

Chronic Trypanosoma cruzi infection potentiates adipose tissue macrophage polarization toward an anti-inflammatory M2 phenotype and contributes to diabetes progression in a diet-induced obesity model

Chronic obesity and Chagas disease (caused by the protozoan Trypanosoma cruzi) represent serious public health concerns. The interrelation between parasite infection, adipose tissue, immune system and metabolism in an obesogenic context, has not been entirely explored. A novel diet-induced obesity model (DIO) was developed in C57BL/6 wild type mice to examine the effect of chronic infection (DIO+I) on metabolic parameters and on obesity-related disorders. Dyslipidemia, hyperleptinemia, and cardiac/hepatic steatosis were strongly developed in DIO mice. Strikingly, although these metabolic alterations were collectively improved by infection, plasmatic apoB100 levels remain significantly increased in DIO+I, suggesting the presence of pro-atherogenic small and dense LDL particles. Moreover, acute insulin resistance followed by chronic hyperglycemia with hypoinsulinemia was found, evidencing an infection-related-diabetes progression. These lipid and glucose metabolic changes seemed to be highly dependent on TLR4 expression since TLR4−/− mice were protected from obesity and its complications. Notably, chronic infection promoted a strong increase in MCP-1 producing macrophages with a M2 (F4/80+CD11c-CD206+) phenotype associated to oxidative stress in visceral adipose tissue of DIO+I mice. Importantly, infection reduced lipid content but intensified inflammatory infiltrates in target tissues. Thus, parasite persistence in an obesogenic environment and the resulting host immunometabolic dysregulation may contribute to diabetes/atherosclerosis progression.

Trypanosoma cruzi, the causative agent of Chagas disease, modulates interleukin-6-induced STAT3 phosphorylation via gp130 cleavage in different host cells

Interleukin-6 mediates host defense and cell survival mainly through the activation of the transcription factor STAT3 via the glycoprotein gp130, a shared signal-transducing receptor for several IL-6-type cytokines. We have reported that the cardiotrophic parasite Trypanosoma cruzi protects murine cardiomyocytes from apoptosis. In agreement, an intense induction of the anti-apoptotic factor Bcl-2 is found in cardiac fibers during the acute phase of infection, establishing a higher threshold against apoptosis. We report here that inactive cruzipain, the main cysteine protease secreted by the parasite, specifically triggered TLR2 and the subsequent release of IL-6, which acted as an essential anti-apoptotic factor for cardiomyocyte cultures. Although comparable IL-6 levels were found under active cruzipain stimulation, starved cardiac cell monolayers could not be rescued from apoptosis. Moreover, cardiomyocytes treated with active cruzipain completely abrogated the STAT3 phosphorylation and nuclear translocation induced by recombinant IL-6. This inhibition was also observed on splenocytes, but it was reverted when the enzyme was complexed with chagasin, a parasite cysteine protease inhibitor. Furthermore, the inhibition of IL-6-induced p-STAT3 was evidenced in spleen cells stimulated with pre-activated supernatants derived from trypomastigotes. To account for these observations, we found that cruzipain enzymatically cleaved recombinant gp130 ectodomain, and induced the release of membrane-distal N-terminal domain of this receptor on human peripheral blood mononuclear cells. These results demonstrate, for the first time, that the parasite may modify the IL-6-induced response through the modulation of its cysteine protease activity, suggesting that specific inhibitors may help to improve the immune cell activation and cardioprotective effects.

CD73 Inhibition Shifts Cardiac Macrophage Polarization toward a Microbicidal Phenotype and Ameliorates the Outcome of Experimental Chagas Cardiomyopathy

Increasing evidence demonstrates that generation of extracellular adenosine from ATP, which is hydrolyzed by the CD39/CD73 enzyme pair, attenuates the inflammatory response and deactivates macrophage antimicrobial mechanisms. Although CD73 is emerging as a critical pathway and therapeutic target in cardiovascular disorders, the involvement of this ectonucleotidase during myocardial infection has not been explored. Using a murine model of infection with Trypanosoma cruzi, the causal agent of Chagas cardiomyopathy, we observed a sudden switch from the classical M1 macrophage (microbicidal) phenotype toward an alternative M2 (repairing/anti-inflammatory) phenotype that occurred within the myocardium very shortly after BALB/c mice infection. The observed shift in M1/M2 rate correlated with the cardiac cytokine milieu. Considering that parasite persistence within myocardium is a necessary and sufficient condition for the development of the chronic myocarditis, we hypothesized that CD73 activity may counteract cardiac macrophage microbicidal polarization, rendering the local immune response less effective. In fact, a transient treatment with a specific CD73 inhibitor (adenosine 5′-α,β-methylene-diphosphate) enhanced the microbicidal M1 subset predominance, diminished IL-4– and IL-10–producing CD4+ T cells, promoted a proinflammatory cytokine milieu, and reduced parasite load within the myocardium during the acute phase. As a direct consequence of these events, there was a reduction in serum levels of creatine kinase muscle–brain isoenzyme, a myocardial-specific injury marker, and an improvement in the electrocardiographic characteristics during the chronic phase. Our results demonstrate that this purinergic system drives the myocardial immune response postinfection and harbors a promising potential as a therapeutic target.

Clomipramine and Benznidazole Act Synergistically and Ameliorate the Outcome of Experimental Chagas Disease

ABSTRACT Chagas disease is an important public health problem in Latin America, and its treatment by chemotherapy with benznidazole (BZ) or nifurtimox remains unsatisfactory. In order to design new alternative strategies to improve the current etiological treatments, in the present work, we comprehensively evaluated the in vitro and in vivo anti-Trypanosoma cruzi effects of clomipramine (CMP) (a parasite-trypanothione reductase-specific inhibitor) combined with BZ. In vitro studies, carried out using a checkerboard technique on trypomastigotes (T. cruzi strain Tulahuen), revealed a combination index (CI) of 0.375, indicative of a synergistic effect of the drug combination. This result was correlated with the data obtained in infected BALB/c mice. We observed that during the acute phase (15 days postinfection [dpi]), BZ at 25 mg/kg of body weight/day alone decreased the levels of parasitemia compared with those of the control group, but when BZ was administered with CMP, the drug combination completely suppressed the parasitemia due to the observed synergistic effect. Furthermore, in the chronic phase (90 dpi), mice treated with both drugs showed less heart damage as assessed by the histopathological analysis, index of myocardial inflammation, and levels of heart injury biochemical markers than mice treated with BZ alone at the reference dose (100 mg/kg/day). Collectively, these data support the notion that CMP combined with low doses of BZ diminishes cardiac damage and inflammation during the chronic phase of cardiomyopathy. The synergistic activity of BZ-CMP clearly suggests a potential drug combination for Chagas disease treatment, which would allow a reduction of the effective dose of BZ and an increase in therapeutic safety.

IL-6 Improves the Nitric Oxide-Induced Cytotoxic CD8+ T Cell Dysfunction in Human Chagas Disease

Reactive oxygen and nitrogen species are important microbicidal agents and are also involved in lymphocyte unresponsiveness during experimental infections. Many of the biological effects attributed to nitric oxide are mediated by peroxynitrites, which induce the nitration of immune cells, among others. Our group has demonstrated that nitric oxide is involved in the suppressive activity of myeloid-derived suppressor cells in Trypanosoma cruzi-infected mice, with a higher number of CD8+ T cells suffering surface-nitration compared to uninfected controls. Studying the functional and phenotypic features of peripheral CD8+ T cells from chagasic patients and human cells experimentally infected with T. cruzi, we found that different regulatory mechanisms impaired the effector functions of T cytotoxic population from seropositive patients. Peripheral leukocytes from chagasic patients showed increased nitric oxide production concomitant with increased tyrosine nitration of CD8+ T cells. Additionally, this cytotoxic population exhibited increased apoptotic rate, loss of the TCRζ-chain, and lower levels of CD107a, a marker of degranulation. Strikingly, IL-6 stimulation of in vitro-infected peripheral blood mononuclear cells obtained from healthy donors, blunted T. cruzi-induced nitration of CD3+CD8+ cells, and increased their survival. Furthermore, the treatment of these cultures with an IL-6 neutralizing antibody increased the percentage of T. cruzi-induced CD8+ T cell nitration and raised the release of nitric oxide. The results suggest that the under-responsiveness of cytotoxic T cell population observed in the setting of long-term constant activation of the immune system could be reverted by the pleiotropic actions of IL-6, since this cytokine improves its survival and effector functions.

New Insights into the Immunobiology of Mononuclear Phagocytic Cells and Their Relevance to the Pathogenesis of Cardiovascular Diseases

Macrophages are the primary immune cells that reside within the myocardium, suggesting that these mononuclear phagocytes are essential in the orchestration of cardiac immunity and homeostasis. Independent of the nature of the injury, the heart triggers leukocyte activation and recruitment. However, inflammation is harmful to this vital terminally differentiated organ with extremely poor regenerative capacity. As such, cardiac tissue has evolved particular strategies to increase the stress tolerance and minimize the impact of inflammation. In this sense, growing evidences show that mononuclear phagocytic cells are particularly dynamic during cardiac inflammation or infection and would actively participate in tissue repair and functional recovery. They respond to soluble mediators such as metabolites or cytokines, which play central roles in the timing of the intrinsic cardiac stress response. During myocardial infarction two distinct phases of monocyte influx have been identified. Upon infarction, the heart modulates its chemokine expression profile that sequentially and actively recruits inflammatory monocytes, first, and healing monocytes, later. In the same way, a sudden switch from inflammatory macrophages (with microbicidal effectors) toward anti-inflammatory macrophages occurs within the myocardium very shortly after infection with Trypanosoma cruzi, the causal agent of Chagas cardiomyopathy. While in sterile injury, healing response is necessary to stop tissue damage; during an intracellular infection, the anti-inflammatory milieu in infected hearts would promote microbial persistence. The balance of mononuclear phagocytic cells seems to be also dynamic in atherosclerosis influencing plaque initiation and fate. This review summarizes the participation of mononuclear phagocyte system in cardiovascular diseases, keeping in mind that the immune system evolved to promote the reestablishment of tissue homeostasis following infection/injury, and that the effects of different mediators could modulate the magnitude and quality of the immune response. The knowledge of the effects triggered by diverse mediators would serve to identify new therapeutic targets in different cardiovascular pathologies.

APP/Go protein Gβγ-complex signaling mediates Aβ degeneration and cognitive impairment in Alzheimer's disease models

Deposition of amyloid-β (Aβ), the proteolytic product of the amyloid precursor protein (APP), might cause neurodegeneration and cognitive decline in Alzheimers disease (AD). However, the direct involvement of APP in the mechanism of Aβ-induced degeneration in AD remains on debate. Here, we analyzed the interaction of APP with heterotrimeric Go protein in primary hippocampal cultures and found that Aβ deposition dramatically enhanced APP-Go protein interaction in dystrophic neurites. APP overexpression rendered neurons vulnerable to Aβ toxicity by a mechanism that required Go-Gβγ complex signaling and p38-mitogen-activated protein kinase activation. Gallein, a selective pharmacological inhibitor of Gβγ complex, inhibited Aβ-induced dendritic and axonal dystrophy, abnormal tau phosphorylation, synaptic loss, and neuronal cell death in hippocampal neurons expressing endogenous protein levels. In the 3xTg-AD mice, intrahippocampal application of gallein reversed memory impairment associated with early Aβ pathology. Our data provide further evidence for the involvement of APP/Go protein in Aβ-induced degeneration and reveal that Gβγ complex is a signaling target potentially relevant for developing therapies for halting Aβ degeneration in AD.

Multi-kinetic release of benznidazole-loaded multiparticulate drug delivery systems based on polymethacrylate interpolyelectrolyte complexes

&NA; Interpolyelectrolyte complexes (IPEC) formulated as multiparticulate drug delivery systems (MDDS) are interesting carriers to improve drug’ performance. Benznidazole (BZ) is the first–line drug for Chagas treatment; however, it presents side effects and toxicity, conditioning its efficacy and safety. The goal of this work was to obtain novel MDDS composed by IPEC based on different polymethacrylate carriers loaded with BZ and to investigate in vitro drug delivery performance for oral administration. Physicochemical characterizations were studied and preclinical studies in a murine model of acute Chagas disease were also performed. The MDDS composed by BZ‐loaded IPEC based on polymethacrylates were obtained by casting solvent followed by wet granulation methods with yields >83%. FT‐IR demonstrated ionic interaction between the polyelectrolytes. Confocal microscopy, DSC and PXRD revealed a fraction uniformly distributed of free BZ on the multiparticles. The rheological evaluation of the MDDS showed adequate flow features for their formulation in hard gelatin‐capsules. The type and composition of IPEC conditioned the modulation of BZ release and fluid uptake results. MDDS based on more hydrophylic Eudragit® showed very fast dissolution (Q15min > 85%), while an extended release (Q120min ≤ 40%) for the hydrophobic ones was observed. Capsules containing a combination of two MDDS with different release profile of BZ showed promising properties to improve Chagas disease pharmacotherapy in the preliminary in vivo assay performed, in which the BZ‐loaded MDDS exhibited efficacy to reduce parasitemia, while decreasing the levels of liver injury markers in comparison to BZ conventional treatment. Multi‐kinetic BZ delivery systems developed are interesting pharmaceutical alternatives to improve the treatment of Chagas disease. Graphical abstract Figure. No Caption available.