Aparecida Donizette Malvezi

Inhibition of cyclooxygenase-1 and cyclooxygenase-2 impairs Trypanosoma cruzi entry into cardiac cells and promotes differential modulation of the inflammatory response.

ABSTRACT The intracellular protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a serious disorder that affects millions of people in Latin America. Cell invasion by T. cruzi and its intracellular replication are essential to the parasites life cycle and for the development of Chagas disease. Here, we present evidence suggesting the involvement of the hosts cyclooxygenase (COX) enzymes during T. cruzi invasion. Pharmacological antagonists for COX-1 (aspirin) and COX-2 (celecoxib) caused marked inhibition of T. cruzi infection when rat cardiac cells were pretreated with these nonsteroidal anti-inflammatory drugs (NSAIDs) for 60 min at 37°C before inoculation. This inhibition was associated with an increase in the production of NO and interleukin-1β and decreased production of transforming growth factor β (TGF-β) by cells. Taken together, these results indicate that COX-1 more than COX-2 is involved in the regulation of anti-T. cruzi activity in cardiac cells, and they provide a better understanding of the influence of TGF-β-interfering therapies on the innate inflammatory response to T. cruzi infection and may represent a very pertinent target for new therapeutic treatments of Chagas disease.

Aspirin Modulates Innate Inflammatory Response and Inhibits the Entry of Trypanosoma cruzi in Mouse Peritoneal Macrophages

The intracellular protozoan parasite Trypanosoma cruzi causes Chagas disease, a serious disorder that affects millions of people in Latin America. Cell invasion by T. cruzi and its intracellular replication are essential to the parasites life cycle and for the development of Chagas disease. Here, we present evidence suggesting the involvement of the hosts cyclooxygenase (COX) enzyme during T. cruzi invasion. Pharmacological antagonist for COX-1, aspirin (ASA), caused marked inhibition of T. cruzi infection when peritoneal macrophages were pretreated with ASA for 30 min at 37°C before inoculation. This inhibition was associated with increased production of IL-1β and nitric oxide (NO∙) by macrophages. The treatment of macrophages with either NOS inhibitors or prostaglandin E2 (PGE2) restored the invasive action of T. cruzi in macrophages previously treated with ASA. Lipoxin ALX-receptor antagonist Boc2 reversed the inhibitory effect of ASA on trypomastigote invasion. Our results indicate that PGE2, NO∙, and lipoxins are involved in the regulation of anti-T. cruzi activity by macrophages, providing a better understanding of the role of prostaglandins in innate inflammatory response to T. cruzi infection as well as adding a new perspective to specific immune interventions.

Oral Exposure to Phytomonas serpens Attenuates Thrombocytopenia and Leukopenia during Acute Infection with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi, the agent of Chagas disease, rapidly develop anemia and thrombocytopenia. These effects are partially promoted by the parasite trans-sialidase (TS), which is shed in the blood and depletes sialic acid from the platelets, inducing accelerated platelet clearance and causing thrombocytopenia during the acute phase of disease. Here, we demonstrate that oral immunization of C57BL/6 mice with Phytomonas serpens, a phytoflagellate parasite that shares common antigens with T. cruzi but has no TS activity, reduces parasite burden and prevents thrombocytopenia and leukopenia. Immunization also reduces platelet loss after intraperitoneal injection of TS. In addition, passive transfer of immune sera raised in mice against P. serpens prevented platelet clearance. Thus, oral exposure to P. serpens attenuates the progression of thrombocytopenia induced by TS from T. cruzi. These findings are not only important for the understanding of the pathogenesis of T. cruzi infection but also for developing novel approaches of intervention in Chagas disease.

Nitric oxide-releasing indomethacin enhances susceptibility to Trypanosoma cruzi infection acting in the cell invasion and oxidative stress associated with anemia

Trypanosoma cruzi is the causative agent of Chagas disease. Approximately 8 million people are thought to be affected with this disease worldwide. T. cruzi infection causes an intense inflammatory response, which is critical for the control of parasite proliferation and disease development. Nitric oxide-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are an emergent class of pharmaceutical derivatives with promising utility as chemopreventive agents. In this study, we investigated the effect of NO-indomethacin on parasite burden, cell invasion, and oxidative stress in erythrocytes during the acute phase of infection. NO-indomethacin was dissolved in dimethyl formamide followed by i.p. administration of 50 ppm into mice 30 min after infection with 5×10(3) blood trypomastigote forms (Y strain). The drug was administered every day until the animals died. Control animals received 100 μL of drug vehicle via the same route. Within the NO-indomethacin-treatment group, parasitemia and mortality (100%) were higher and oxidative stress in erythrocytes, anemia, and entry of parasites into macrophages were significantly greater than that seen in controls. Increase in the entry and survival of intracellular T. cruzi was associated with inhibition of nitric oxide production by macrophages treated with NO-indomethacin (2.5 μM). The results of this study provide strong evidence that NO-NSAIDs potently inhibit nitric oxide production, suggesting that NO-NSAID-based therapies against infections would be difficult to design and would require caution.

Fish oil supplementation benefits the murine host during the acute phase of a parasitic infection from Trypanosoma cruzi

Long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) are known to modulate a variety of immune cell functions. On occasion, this has led to diminished host resistance to certain viral and bacterial infections. Little is known about the impact of n-3 PUFA on host resistance to parasitic infection, however, based on results from a small study conducted more than two decades ago, we hypothesized that providing mice LC n-3 PUFA will diminish host resistance to Trypanosoma cruzi, the parasitic pathogen responsible for Chagas disease. To investigate this, C57BL/6 mice were supplemented by gavage (0.6% v/w) with phosphate-buffered saline, corn oil (CO), or menhaden fish oil (FO, a fat source rich in LC n-3 PUFA) for 15 days prior to T cruzi (Y strain) challenge and throughout the acute phase of infection. FO supplementation was associated with a transient 2-fold greater peak of blood parasitemia at 7 days postinfection (dpi), whereas subsequent cardiac parasitemia was ~60% lower at 12 dpi. FO treatment also ameliorated the leukopenia and thrombocytopenia observed in the early stages of a T cruzi infection. FO supplementation reduced circulating and cardiac nitric oxide at 7 and 12 dpi, respectively. FO supplementation altered ex vivo prostaglandin E2 and cytokine and chemokine production by splenocytes isolated from uninfected and infected mice. Overall, our results suggest that oral administration of LC n-3 PUFA from FO can have beneficial effects on the host in the early course of a T cruzi infection.

Response to Trypanosoma cruzi by human blood cells enriched with Dentritic Cells is controlled by Cyclooxygenase-2 Pathway

Chagas disease (Cd) or American human trypanosomiasis is caused by Trypanosoma cruzi and affects ~7 million people, mostly in Latin America. The infective trypomastigote forms of the parasite can invade several human blood cell populations, including monocytes and dendritic cells (DC). Although these cells display a wide functional diversity, their interactions with T. cruzi via cyclooxygenase (COX) and cyclic adenosine monophosphate (cAMP) dependent pathways have not been analyzed. To exploiting this mechanism, DC-enriched peripheral human blood mononuclear cell populations (DC-PBMC) were used as our model. Our results showed that the treatment of these cell populations with celecoxib (CEL), a cyclooxygenase-2 selective inhibitor or SQ 22,536, an adenilate cyclase inhibitor, significantly caused marked inhibition of T. cruzi infection. In contrast, aspirin (ASA, a non-selective COX-1 and COX-2 inhibitor) treatment did not inhibit the infection of the cells by the parasite and was independent of nitric oxide (NO) production. The expression of co-stimulatory molecules CD80 and CD86 were similar on cells treated or not with both COX-inhibitors. The infection stimulated the release of TNF-α, IL-1β, IL-6, IL-8, and IL-10 production by infected cells. Treatment with ASA or CEL did not affect TNF-α, IL-6, IL-8, IL-10, and NO production by infected cells, but increased IL-1β production by them. Our results suggest a key role of COX-2 and cAMP pathways in T. cruzi invasion process of human blood cells and these pathways may represent targets of new therapeutic options for Cd.

Extracellular Vesicles Shed By Trypanosoma cruzi Potentiate Infection and Elicit Lipid Body Formation and PGE2 Production in Murine Macrophages

During the onset of Trypanosoma cruzi infection, an effective immune response is necessary to control parasite replication and ensure host survival. Macrophages have a central role in innate immunity, acting as an important trypanocidal cell and triggering the adaptive immune response through antigen presentation and cytokine production. However, T. cruzi displays immune evasion mechanisms that allow infection and replication in macrophages, favoring its chronic persistence. One potential mechanism is the release of T. cruzi strain Y extracellular vesicle (EV Y), which participate in intracellular communication by carrying functional molecules that signal host cells and can modulate the immune response. The present work aimed to evaluate immune modulation by EV Y in C57BL/6 mice, a prototype resistant to infection by T. cruzi strain Y, and the effects of direct EV Y stimulation of macrophages in vitro. EV Y inoculation in mice prior to T. cruzi infection resulted in increased parasitemia, elevated cardiac parasitism, decreased plasma nitric oxide (NO), reduced NO production by spleen cells, and modulation of cytokine production, with a reduction in TNF-α in plasma and decreased production of TNF-α and IL-6 by spleen cells from infected animals. In vitro assays using bone marrow-derived macrophages showed that stimulation with EV Y prior to infection by T. cruzi increased the parasite internalization rate and release of infective trypomastigotes by these cells. In this same scenario, EV Y induced lipid body formation and prostaglandin E2 (PGE2) production by macrophages even in the absence of T. cruzi. In infected macrophages, EV Y decreased production of PGE2 and cytokines TNF-α and IL-6 24 h after infection. These results suggest that EV Y modulates the host response in favor of the parasite and indicates a role for lipid bodies and PGE2 in immune modulation exerted by EVs.