Nora Goren

15-deoxy-Delta12,14 prostaglandin GJ2 but not rosiglitazone regulates metalloproteinase 9, NOS-2, and cyclooxygenase 2 expression and functions by peroxisome proliferator-activated receptor gamma-dependent and -independent mechanisms in cardiac cells.

Sepsis or endotoxemia produced by LPS followed by hypotension and multiorganic failure may lead to cardiac dysfunction contributing to mortality. Cardiac failure is usually associated to activation of nuclear factor &kgr;B (NF-&kgr;B) and mitogen-activated protein kinase (MAPK), which play an important role in proinflammatory enzymes expression. It has been shown that 15-deoxy-&Dgr;12,14 prostaglandin J2 (15dPGJ2) can repress the inflammatory response by means of peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;)-dependent and -independent mechanisms. However, its precise role in heart is poorly understood. In the present study, mouse neonatal cardiomyocytes were isolated and stimulated with LPS to investigate the role of PPAR&ggr;-specific ligands 15dPGJ2 and rosiglitazone on cardiac inflammatory response. Inducible NO synthase, cyclooxygenase 2, and metalloproteinase 9 mRNA levels, protein expression, and activity were inhibited with 15dPGJ2 but not by rosiglitazone. Peroxisome proliferator-activated receptor &ggr; antagonist, GW9662, prevented all these 15dPGJ2 actions. To go inside the mechanisms by which 15dPGJ2 exerts inhibitory effects, cells were preincubated with specific chemical inhibitors of NF-&kgr;B and p38 MAPK, and we found that these signaling cascades are implicated in 15dPGJ2 action as well as PPAR&ggr;. These results suggest that only the natural PPAR&ggr; ligand, 15dPGJ2, but not the synthetic one, rosiglitazone, regulates the inflammatory response by inhibition of inducible NO synthase, cyclooxygenase 2, and metalloproteinase 9 expression. Moreover, our results offer an additional 15dPGJ2 mechanism of action, despite PPAR&ggr;, showing NF-&kgr;B and p38 MAPK participation.

Treatment in vitro with PPARα and PPARγ ligands drives M1-to-M2 polarization of macrophages from T. cruzi-infected mice.

Trypanosoma cruzi, the etiological agent of Chagas disease, induces a persistent inflammatory response. Macrophages are a first line cell phenotype involved in the clearance of infection. Upon parasite uptake, these cells increase inflammatory mediators like NO, TNF-α, IL-1β and IL-6, leading to parasite killing. Although desired, inflammatory response perpetuation and exacerbation may lead to tissue damage. Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent nuclear transcription factors that, besides regulating lipid and carbohydrate metabolism, have a significant anti-inflammatory effect. This is mediated through the interaction of the receptors with their ligands. PPARγ, one of the PPAR isoforms, has been implicated in macrophage polarization from M1, the classically activated phenotype, to M2, the alternatively activated phenotype, in different models of metabolic disorders and infection. In this study, we show for the first time that, besides PPARγ, PPARα is also involved in the in vitro polarization of macrophages isolated from T. cruzi-infected mice. Polarization was evidenced by a decrease in the expression of NOS2 and proinflammatory cytokines and the increase in M2 markers like Arginase I, Ym1, mannose receptor and TGF-β. Besides, macrophage phagocytic activity was significantly enhanced, leading to increased parasite load. We suggest that modulation of the inflammatory response by both PPARs might be due, at least in part, to a change in the profile of inflammatory macrophages. The potential use of PPAR agonists as modulators of overt inflammatory response during the course of Chagas disease deserves further investigation.

Inducible nitric oxide synthase-mediated proliferation of a T lymphoma cell line

Nitric oxide (NO)-derived from T lymphocytes in an autocrine fashion can modulate events in the cell. However, the exact role of NO on the control of lymphocyte growth is controversial since both stimulation and inhibition have been demonstrated. Nitric oxide synthase (NOS) activity in normal and tumor T lymphocyte proliferation was studied here. Resting normal T lymphocytes displayed low levels of NOS activity that were slightly increased upon mitogenic stimulation. In contrast, BW5147 T lymphoma cells displayed higher basal levels than normal T lymphocytes that were significantly augmented when induced to proliferate. This activity was slightly modified in the presence of the calcium chelator EGTA and was blocked by competitive and irreversible NOS inhibitors, as well as by selective blockers of iNOS. Furthermore, tumor but not normal cell proliferation was impaired by NOS and iNOS blockers, while a calcium blocker only affected normal cell growth. iNOS expression, both at the protein and at the mRNA levels, was demonstrated on growing BW5147 cells but not on arrested tumor or normal lymphocytes. The contribution of iNOS to sustained proliferation of tumor cells is discussed.

Involvement of intestinal inducible nitric oxide synthase (iNOS) in the early stages of murine salmonellosis.

Local induction of inducible nitric oxide synthase (iNOS) and apoptosis was examined in the intestine of mice infected with virulent Salmonella enterica serovar Enteritidis 5694 (S. enteritidis) and its attenuated derivative mutant E/1/3. Both, intestinal iNOS mRNA expression and iNOS activity showed a peak at 4 h only in animals receiving the virulent S. enteritidis. Aminoguanidine treatment abrogated intestinal epithelial damage produced by virulent S. enteritidis and diminished apoptosis at the tips of the villi. Unlike the virulent strain, mutant E/1/3 induced massive iNOS expression in Peyers patches, these findings may be related to its protective capacity. Our results suggest that intestinal iNOS participates in the early response to intestinal infection and that the final effect depends on the nature of the insult.

Role of PPARs in Trypanosoma cruzi Infection: Implications for Chagas Disease Therapy

Chagas disease, which is caused by Trypanosoma cruzi (T. cruzi), remains a substantial public health concern and an important cause of morbidity and mortality in Latin America. T. cruzi infection causes an intense inflammatory response in diverse tissues by triggering local expression of inflammatory mediators, which results in the upregulation of the levels of cytokines and chemokines, and important cardiac alterations in the host, being one of the most characteristic damages of Chagas disease. Therefore, controlling the inflammatory reaction becomes critical for the control of the proliferation of the parasite and of the evolution of Chagas disease. The nuclear receptors known as peroxisome proliferator-activated receptors (PPARs) have emerged as key regulators of lipid metabolism and inflammation. The precise role of PPAR ligands in T. cruzi infection or in Chagas disease is poorly understood. This review summarizes our knowledge about T. cruzi infection as well as about the activation of PPARs and the potential role of their ligands in the resolution of inflammation, with the aim to address a new pharmacological approach to improve the host health.

Salmonella enterica serovar Enteritidis dam mutant induces low NOS-2 and COX-2 expression in macrophages via attenuation of MAPK and NF-κB pathways

Although dam mutants of Salmonella have been proposed as live vaccines, their capacity to trigger cell inflammatory cascades has not been fully elucidated. We investigated in detail the ability of Salmonella enterica dam mutant to activate the signalling pathways of the inflammatory response in RAW 264.7 cells. Apoptosis in macrophages treated with Salmonella dam mutant was low. Similarly, the expression of both NOS-2 and COX-2 and subsequently the production of NO and PGE(2) was significantly reduced. Also, Salmonella dam mutant induced an attenuated activation of the inflammatory signalling pathway as indicated by the reduced degradation of IkappaBalpha and IkappaBbeta and the low IkappaBalpha phosphorylation found. In addition, translocation of p65 to the nucleus was notably impaired and the amount of phosphorylated p44, p42 and p38 MAPKs was clearly reduced in extracts from dam-infected macrophages. These results indicate that the lack of ERK and p38 phosphorylation at the proper time in dam-infected cells notably reduces the engagement of subsequent signalling pathways involved in the full activation of NF-kappaB in response to infection. Taken together, these results suggest that Salmonella activation of both signalling cascades in the inflammatory response is a mechanism requiring Dam protein participation.

Low-dose benznidazole treatment results in parasite clearance and attenuates heart inflammatory reaction in an experimental model of infection with a highly virulent Trypanosoma cruzi strain

Chagas disease, caused by Trypanosoma cruzi, is the main cause of dilated cardiomyopathy in the Americas. Antiparasitic treatment mostly relies on benznidazole (Bzl) due to Nifurtimox shortage or unavailability. Both induce adverse drug effects (ADE) of varied severity in many patients, leading to treatment discontinuation or abandonment. Since dosage may influence ADE, we aimed to assess Bzl efficacy in terms of parasiticidal and anti-inflammatory activity, using doses lower than those previously reported. BALB/c mice infected with the T. cruzi RA strain were treated with different doses of Bzl. Parasitaemia, mortality and weight change were assessed. Parasite load, tissue infiltrates and inflammatory mediators were studied in the heart. Serum creatine kinase (CK) activity was determined as a marker of heart damage. The infection-independent anti-inflammatory properties of Bzl were studied in an in vitro model of LPS-treated cardiomyocyte culture. Treatment with 25 mg/kg/day Bzl turned negative the parasitological parameters, induced a significant decrease in IL-1β, IL-6 and NOS2 in the heart and CK activity in serum, to normal levels. No mortality was observed in infected treated mice. Primary cultured cardiomyocytes treated with Bzl showed that inflammatory mediators were reduced via inhibition of the NF-κB pathway. A Bzl dose lower than that previously reported for treatment of experimental Chagas disease exerts adequate antiparasitic and anti-inflammatory effects leading to parasite clearance and tissue healing. This may be relevant to reassess the dose currently used for the treatment of human Chagas disease, aiming to minimize ADE.

Effect of histamine in autoimmune myocarditis mice

The contractile effect of histamine, as well as the H1 receptor population and H2 receptor-mediated cAMP production, were measured in cardiac tissue from control normal and autoimmune myocarditis mice. Histamine triggered positive chronotropy and negative inotropy at high concentrations in both control and autoimmune auricles, H2 receptors being the most important mediator of these responses. In contrast, in atria from autoimmune myocarditis mice, histamine at lower concentrations caused positive inotropy and negative chronotropy. These effects, not verified in the normal control atria, are mediated by H1 receptors. The expression of H2 and H1 receptors mediating the cardiac response to histamine was evaluated through histamine-stimulated cAMP level and binding of [3H] mephyramine, respectively. Both control and autoimmune myocardium were able to increase cAMP levels, an effect that was inhibited by H2 antagonist drug. The amount of cAMP was significantly higher in control myocardium than in those from autoimmune ones. Saturable binding of [3H] mephyramine occurs in autoimmune myocardium, with distinct high and low affinity binding sites. In control myocardium non-saturable binding was detected. These results suggest that H1 and H2 receptors coexist in heart from autoimmune myocarditis mice, whereas only H2 receptors are present in myocardium from control mice. The presence of H1 receptors in autoimmune myocardium could be an important factor in the regulation of its physiological behaviour.

Increases in cyclic AMP levels couple to H1 receptors in atria from autoimmune myocarditis mice

We have previously shown that myocardium from experimental autoimmune myocarditis expresses H1 receptors not present in normal mice heart. ThEA acting via H1 receptors, augments cyclic AMP production in atria from autoimmune myocarditis mice without any effect on atria from control mice. Addition of mepyramine before ThEA caused cyclic AMP levels to fall to a level similar to basal, confirming the H1 receptor participation. Histamine at low concentrations mimicked the ThEA action on H1 receptor-stimulation of cyclic AMP production by autoimmune myocardium. The fact that the inhibition of phospholipase C blocked the cyclic AMP stimulation by ThEA, supports the assumption that this action is secondary to receptor-mediated hydrolysis of phosphoinositides, generating some oxidative metabolites (IP3-DAG), which in turn may be responsible for the cyclic AMP effect. So, the inhibition of protein kinase C and calcium/calmodulin partially prevented the stimulatory action of ThEA on cyclic AMP levels in autoimmune myocardium, suggesting that both pathways are implicated in this effect. Data shows that the stimulation of H1 receptors by specific agonist in atria from autoimmune myocarditis mice, augments the cyclic AMP, requiring the hydrolysis of phosphoinositide cycle. The role of this cyclic AMP augmentation in myocardium from autoimmune myocarditis mice, will provide a basis to assess the role of this second messenger as an important factor in the regulation and/or modulation of the physiological behaviour of the heart in the course of autoimmune myocarditis.

Treatment with a New Peroxisome Proliferator-Activated Receptor Gamma Agonist, Pyridinecarboxylic Acid Derivative, Increases Angiogenesis and Reduces Inflammatory Mediators in the Heart of Trypanosoma cruzi-Infected Mice

Trypanosoma cruzi infection induces an intense inflammatory response in diverse host tissues. The immune response and the microvascular abnormalities associated with infection are crucial aspects in the generation of heart damage in Chagas disease. Upon parasite uptake, macrophages, which are involved in the clearance of infection, increase inflammatory mediators, leading to parasite killing. The exacerbation of the inflammatory response may lead to tissue damage. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent nuclear transcription factor that exerts important anti-inflammatory effects and is involved in improving endothelial functions and proangiogenic capacities. In this study, we evaluated the intermolecular interaction between PPARγ and a new synthetic PPARγ ligand, HP24, using virtual docking. Also, we showed that early treatment with HP24, decreases the expression of NOS2, a pro-inflammatory mediator, and stimulates proangiogenic mediators (vascular endothelial growth factor A, CD31, and Arginase I) both in macrophages and in the heart of T. cruzi-infected mice. Moreover, HP24 reduces the inflammatory response, cardiac fibrosis and the levels of inflammatory cytokines (TNF-α, interleukin 6) released by macrophages of T. cruzi-infected mice. We consider that PPARγ agonists might be useful as coadjuvants of the antiparasitic treatment of Chagas disease, to delay, reverse, or preclude the onset of heart damage.