HIF-1α and CD73 expression in cardiac leukocytes correlates with the severity of myocarditis in end-stage Chagas disease patients.

Chronic Chagas cardiomyopathy is the main infectious myocarditis worldwide. Almost 30% of Trypanosoma cruzi infected individuals develop slow and progressive myocarditis that leads to ventricular dilation and heart failure. Heart transplantation is an established, valuable therapeutic option for end-stage Chagas disease patients. Although the pathophysiology of Chagas disease has been addressed for decades by numerous groups, the cardiac immunologic mechanisms involved in the progression of clinical manifestation are still unknown. Growing evidence demonstrates that hypoxia-inducible factor (HIF)-1α plays indispensable roles in driving immune response by triggering the expression of CD73 purinergic ecto-enzyme. Purinergic system controls the duration and magnitude of purine signals directed to modulate immune cells through the conversion of extracellular ATP (microbicide/proinflammatory) to the immunoregulatory metabolite adenosine. In the present work, we described that infiltrating leukocytes within cardiac explants from patients with end-stage Chagas cardiomyopathy up-regulated HIF-1α and CD73 expression. Moreover, the number of HIF-1α+ and CD73+ leukocytes positively correlated with the myocarditis severity and the local parasite load. Furthermore, we demonstrated a direct relationship between tissue parasite persistence and the influx of immune cells to the infected hearts, which ultimately determine the severity of the myocarditis. These findings provide evidence that CD73-dependent regulatory pathways are locally triggered in the myocardium of patients with end-stage Chagas disease.

Deficiency of CD73 activity promotes protective cardiac immunity against Trypanosoma cruzi infection but permissive environment in visceral adipose tissue.

Damaged cells release the pro-inflammatory signal ATP, which is degraded by the ectonucleotidases CD39 and CD73 to the anti-inflammatory mediator adenosine (ADO). The balance between ATP/ADO is known to determine the outcome of inflammation/infection. However, modulation of the local immune response in different tissues due to changes in the balance of purinergic metabolites has yet to be investigated. Here, we explored the contribution of CD73-derived ADO on the acute immune response against Trypanosoma cruzi parasite, which invades and proliferates within different target tissues. Deficiency of CD73 activity led to an enhanced cardiac microbicidal immune response with an augmented frequency of macrophages with inflammatory phenotype and increased CD8+ T cell effector functions. The increment of local inducible nitric oxide (NO) synthase (iNOS)+ macrophages and the consequent rise of myocardial NO production in association with reduced ADO levels induced protection against T. cruzi infection as observed by the diminished cardiac parasite burden compared to their wild-type (WT) counterpart. Unexpectedly, parasitemia was substantially raised in CD73KO mice in comparison with WT mice, suggesting the existence of tissue reservoir/s outside myocardium. Indeed, CD73KO liver and visceral adipose tissue (VAT) showed increased parasite burden associated with a reduced ATP/ADO ratio and the lack of substantial microbicidal immune response. These data reveal that the purinergic system has a tissue-dependent impact on the host immune response against T. cruzi infection.

Pro-inflammatory monocyte profile in patients with major depressive disorder and suicide behaviour and how ketamine induces anti-inflammatory M2 macrophages by NMDAR and mTOR.

BACKGROUND: Depression is a highly prevalent disorder that is one of the leading causes of disability worldwide. Despite an unknown aetiology, evidence suggests that the innate and adaptive immune systems play a significant role in the development and maintenance of major depressive disorder (MDD). The non-competitive glutamatergic N-methyl-D-aspartate receptor (NMDAR) antagonist, (R,S)-ketamine (ketamine), has demonstrated rapid and robust efficacy as an antidepressant when administered at sub-anaesthetic doses. METHODS: Our goal was to characterize the pro-inflammatory profile of patients with MDD by measuring pro-inflammatory cytokines in plasma and circulating monocyte subsets and to understand how ketamine induces an anti-inflammatory program in monocyte and macrophages in vitro and vivo. FINDING: Our results show that patients with MDD without other comorbidities (N = 33) exhibited significantly higher levels of pro-inflammatory IL-12 and IL-6 in plasma and that these cytokines were associated with increased numbers of non-classical (CD11b+CD16brightCD14neg) monocytes and increased activation state (CD40+CD86+) of classical monocytes in circulation. Remarkably, we have demonstrated that sub-anaesthetic doses of ketamine programs human monocytes into M2c-like macrophages by inducing high levels of CD163 and MERTK with intermediate levels of CD64 and stimulating mTOR-associated gene expression in vitro. The NMDAR antagonist MK-801, but not the α-amino-3­hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonist, NBQX, also polarizes macrophages to an M2c-like phenotype, but this phenotype disappears upon mTOR pathway inhibition. Sub-anaesthetic doses (10 mg/kg) of ketamine administration in mice both promote reduction of circulating classical pro-inflammatory monocytes and increase of alternative M2 macrophage subtypes in the spleen and CNS. INTERPRETATION: Our results suggest an anti-inflammatory property of ketamine that can skew macrophages to an M2-like phenotype, highlighting potential therapeutic implications not only for patients with MDD but also other inflammatory-based diseases. FUNDING: This study was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT-FONCYT).

Isolation and Phenotypic Characterization of Inflammatory Cells from Clinical Samples: Purification of Macrophages from Trypanosoma cruzi-Infected Hearts.

Trypanosoma cruzi, the causal agent of chronic Chagas cardiomyopathy, exhibits an important tropism for cardiac tissue. In consequence, T. cruzi experimental infection represents a unique model to study cardiac macrophage behavior and effector functions during either acute or chronic immune response. In this chapter we describe a protocol to isolate immune cells from T. cruzi-infected murine cardiac tissue and to determine the percentage, absolute number, phenotype, and functionality of monocytes and macrophages by using flow cytometry. Moreover, we describe the parameters to discriminate between resident and infiltrating mononuclear phagocytic cells within infected hearts. The investigations in this field will provide mechanistic insights about the roles of these innate immune cells in the context of a clinically relevant target tissue.

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,ß-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.