The combined action of glycoinositolphospholipid from Trypanosoma cruzi and macrophage migration inhibitory factor increases proinflammatory mediator production by cardiomyocytes and vascular endothelial cells.

Cardiomyopathy is the most serious complication of chronic Chagas disease, caused by infection with the protozoan Trypanosoma cruzi. Exacerbated inflammation of the myocardium constitutes a major pathologic component of the disease. In the myocardial microenvironment, parasite antigens and host inflammatory mediators may aggravate tissue damage. The glycoinositolphospholipid (GIPL) from T. cruzi is an inflammation-eliciting antigen recognized by Toll-like receptor 4 (TLR4), whereas the proinflammatory cytokine macrophage migration inhibitory factor (MIF) promotes progression of chronic Chagas cardiomyopathy. We herein aimed to examine the involvement of GIPL and MIF in molecular mechanisms leading to a pathogenic inflammatory response in HL-1 cardiomyocytes and HMEC microvascular endothelial cells. Immunofluorescence analysis revealed that GIPL enhanced TLR4 expression in both cell types. We found that TLR4/GIPL interaction and MIF activity modulated the arachidonic acid pathway implicated in persistent inflammation. The combination of GIPL at 50 µg/ml and MIF at 50 ng/ml upregulated type 2 cyclooxygenase (COX-2) levels in HL-1 and HMEC cells, in a stronger way than each molecule acting independently. Moreover, increased expression of prostanoid synthases and release of prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) were detected in stimulated cells. Transfection experiments in HL-1 and HMEC cells showed that COX-2 induction was transcriptionally regulated through GIPL-TLR4 engagement and NFκB signaling cascade. (GIPL + MIF)-triggered NFκB activation was markedly attenuated by treatment with 100 µM Fenofibrate, a PPAR-α ligand. Fenofibrate reduced COX-2-dependent generation of bioactive lipids in HL-1 and HMEC cells. In addition, Fenofibrate abolished (GIPL + MIF)-fostered release of NO, IL-1ß, IL-6, TNF-α, and CCL2. The combined actions of GIPL and MIF display potential for amplifying the inflammatory response in myocardium of parasite-infected hosts. Our current findings might help develop more effective measures to ameliorate cardiovascular abnormalities associated with Chagas heart disease.

Benznidazole Anti-Inflammatory Effects in Murine Cardiomyocytes and Macrophages Are Mediated by Class I PI3Kδ.

Benznidazole (Bzl), the drug of choice in many countries for the treatment of Chagas disease, leads to parasite clearance in the early stages of infection and contributes to immunomodulation. In addition to its parasiticidal effect, Bzl inhibits the NF-κB pathway. In this regard, we have previously described that this occurs through IL-10/STAT3/SOCS3 pathway. PI3K pathway is involved in the regulation of the immune system by inhibiting NF-κB pathway through STAT3. In this work, the participation of PI3K in the immunomodulatory effects of Bzl in cardiac and immune cells, the main targets of Chagas disease, was further studied. For that, we use a murine primary cardiomyocyte culture and a monocyte/macrophage cell line (RAW 264.7), stimulated with LPS in presence of LY294002, an inhibitor of PI3K. Under these conditions, Bzl could neither increase SOCS3 expression nor inhibit the NOS2 mRNA expression and the release of NOx, both in cardiomyocytes and macrophages. Macrophages are crucial in the development of Chronic Chagas Cardiomyopathy. Thus, to deepen our understanding of how Bzl acts, the expression profile of M1-M2 macrophage markers was evaluated. Bzl inhibited the release of NOx (M1 marker) and increased the expression of Arginase I (M2 marker) and a negative correlation was found between them. Besides, LPS increased the expression of pro-inflammatory cytokines. Bzl treatment not only inhibited this effect but also increased the expression of typical M2-macrophage markers like Mannose Receptor, TGF-ß, and VEGF-A. Moreover, Bzl increased the expression of PPAR-γ and PPAR-α, known as key regulators of macrophage polarization. PI3K directly regulates M1-to-M2 macrophage polarization. Since p110δ, catalytic subunit of PI3Kδ, is highly expressed in immune cells, experiments were carried out in presence of CAL-101, a specific inhibitor of this subunit. Under this condition, Bzl could neither increase SOCS3 expression nor inhibit NF-κB pathway. Moreover, Bzl not only failed to inhibit the expression of pro-inflammatory cytokines (M1 markers) but also could not increase M2 markers. Taken together these results demonstrate, for the first time, that the anti-inflammatory effect of Bzl depends on PI3K activity in a cell line of murine macrophages and in primary culture of neonatal cardiomyocytes. Furthermore, Bzl-mediated increase expression of M2-macrophage markers involves the participation of the p110δ catalytic subunit of PI3Kδ.

Fenofibrate Increases the Population of Non-Classical Monocytes in Asymptomatic Chagas Disease Patients and Modulates Inflammatory Cytokines in PBMC.

Chronic Chagas disease cardiomyopathy (CCC) is the most important clinical manifestation of infection with Trypanosma cruzi (T. cruzi) due to its frequency and effects on morbidity and mortality. Peripheral blood mononuclear cells (PBMC) infiltrate the tissue and differentiate into inflammatory macrophages. Advances in pathophysiology show that myeloid cell subpopulations contribute to cardiac homeostasis, emerging as possible therapeutic targets. We previously demonstrated that fenofibrate, PPARα agonist, controls inflammation, prevents fibrosis and improves cardiac function in a murine infection model. In this work we investigated the spontaneous release of inflammatory cytokines and chemokines, changes in the frequencies of monocyte subsets, and fenofibrate effects on PBMC of seropositive patients with different clinical stages of Chagas disease. The results show that PBMC from Chagas disease patients display higher levels of IL-12, TGF-ß, IL-6, MCP1, and CCR2 than cells from uninfected individuals (HI), irrespectively of the clinical stage, asymptomatic (Asy) or with Chagas heart disease (CHD). Fenofibrate reduces the levels of pro-inflammatory mediators and CCR2 in both Asy and CHD patients. We found that CHD patients display a significantly higher percentage of classical monocytes in comparison with Asy patients and HI. Besides, Asy patients have a significantly higher percentage of non-classical monocytes than CHD patients or HI. However, no difference in the intermediate monocyte subpopulation was found between groups. Moreover, monocytes from Asy or CHD patients exhibit different responses upon stimulation in vitro with T. cruzi lysates and fenofibrate treatment. Stimulation with T. cruzi significantly increases the percentage of classical monocytes in the Asy group whereas the percentage of intermediate monocytes decreases. Besides, there are no changes in their frequencies in CHD or HI. Notably, stimulation with T. cruzi did not modify the frequency of the non-classical monocytes subpopulation in any of the groups studied. Moreover, fenofibrate treatment of T. cruzi-stimulated cells, increased the frequency of the non-classical subpopulation in Asy patients. Interestingly, fenofibrate restores CCR2 levels but does not modify HLA-DR expression in any groups. In conclusion, our results emphasize a potential role for fenofibrate as a modulator of monocyte subpopulations towards an anti-inflammatory and healing profile in different stages of chronic Chagas disease.

IL-10-Dependent and -Independent Mechanisms Are Involved in the Cardiac Pathology Modulation Mediated by Fenofibrate in an Experimental Model of Chagas Heart Disease.

IL-10 is an anti-inflammatory cytokine that plays a significant role in the modulation of the immune response in many pathological conditions, including infectious diseases. Infection with Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas disease, results in an ongoing inflammatory response that may cause heart dysfunction, ultimately leading to heart failure. Given its infectious and inflammatory nature, in this work we analyzed whether the lack of IL-10 hinders the anti-inflammatory effects of fenofibrate, a PPARα ligand, in a murine model of Chagas heart disease (CHD) using IL-10 knockout (IL-10 KO) mice. Our results show fenofibrate was able to restore the abnormal cardiac function displayed by T. cruzi-infected mice lacking IL-10. Treatment with fenofibrate reduced creatine kinase (CK) levels in sera of IL-10 KO mice infected with T. cruzi. Moreover, although fenofibrate could not modulate the inflammatory infiltrates developing in the heart, it was able to reduce the increased collagen deposition in infected IL-10 KO mice. Regarding pro-inflammatory mediators, the most significant finding was the increase in serum IL-17. These were reduced in IL-10 KO mice upon fenofibrate treatment. In agreement with this, the expression of RORγt was reduced. Infection of IL-10 KO mice increased the expression of YmI, FIZZ and Mannose Receptor (tissue healing markers) that remained unchanged upon treatment with fenofibrate. In conclusion, our work emphasizes the role of anti-inflammatory mechanisms to ameliorate heart function in CHD and shows, for the first time, that fenofibrate attains this through IL-10-dependent and -independent mechanisms.

IL-10/STAT3/SOCS3 Axis Is Involved in the Anti-inflammatory Effect of Benznidazole.

Anti-parasitic treatment for Chagas disease mainly relies on benznidazole, which is virtually the only drug available in the market. Besides its anti-parasitic effects, benznidazole has anti-inflammatory properties. In this work we studied the mechanisms involved in the latter, demonstrating the participation of the IL-10/STAT3/SOCS3 pathway. To achieve this goal, the anti-inflammatory properties of benznidazole were studied using an in vitro model of cardiomyocyte primary culture stimulated with LPS. LPS increased both SOCS3 expression and STAT3 phosphorylation. The addition of benznidazole increased their expression even further. Specific inhibition of STAT3 precluded this effect, suggesting a role for STAT3 in the increase of SOCS3 expression induced by benznidazole. To assess the participation of SOCS3 in the anti-inflammatory effect of benznidazole, we accomplished specific knockdown of SOCS3 with siRNA. Silencing of SOCS3 in cardiomyocytes precluded the inhibitory effects of benznidazole on TNF-α, IL-6, iNOS expression and NO release. Moreover, in the absence of SOCS3, benznidazole could neither prevent IKK phosphorylation nor IκBα degradation, supporting the notion that SOCS3 is required for the benznidazole-mediated inhibition of the NF-κB pathway. Previously, we demonstrated that IL-10 increases the expression of SOCS3 in cultured cardiomyocytes. Here, we found that benznidazole shows a trend to increased IL-10 expression. To evaluate whether benznidazole increased SOCS3 in an IL-10-dependent manner, cardiomyocytes from IL-10 knockout mice were pre-treated with benznidazole and stimulated with LPS. Benznidazole neither inhibited NO release nor avoid IKK phosphorylation or IκBα degradation, showing that IL-10 is required for benznidazole-mediated inhibition of NF-κB. Moreover, exogenous addition of IL-10 to IL-10 knockout cardiomyocytes restored the inhibitory effect of benznidazole on NO release. The results reported herein show, for the first time, that the IL-10/STAT3/SOCS3 axis is involved in the anti-inflammatory effects of benznidazole. These findings may add up to new therapeutic strategies for chronic Chagas disease given its inflammatory nature.

Treatment with Fenofibrate plus a low dose of Benznidazole attenuates cardiac dysfunction in experimental Chagas disease.

Trypanosoma cruzi induces serious cardiac alterations during the chronic infection. Intense inflammatory response observed from the beginning of infection, is critical for the control of parasite proliferation and evolution of Chagas disease. Peroxisome proliferator-activated receptors (PPAR)-α, are known to modulate inflammation. In this study we investigated whether a PPAR-α agonist, Fenofibrate, improves cardiac function and inflammatory parameters in a murine model of T. cruzi infection. BALB/c mice were sequentially infected with two T. cruzi strains of different genetic background. Benznidazole, commonly used as trypanocidal drug, cleared parasites but did not preclude cardiac pathology, resembling what is found in human chronic chagasic cardiomyopathy. Fenofibrate treatment restored to normal values the ejection and shortening fractions, left ventricular end-diastolic, left ventricular end-systolic diameter, and isovolumic relaxation time. Moreover, it reduced cardiac inflammation and fibrosis, decreased the expression of pro-inflammatory (IL-6, TNF-α and NOS2) and heart remodeling mediators (MMP-9 and CTGF), and reduced serum creatine kinase activity. The fact that Fenofibrate partially inhibited NOS2 expression and NO release in the presence of a PPAR-α non-competitive inhibitor, suggested it also acted through PPAR-α-independent pathways. Since IκBα cytosolic degradation was inhibited by Fenofibrate, it can be concluded that the NFκB pathway has a role in its effects. Thus, we demonstrate that Fenofibrate acts through PPAR-α-dependent and -independent pathways. Our study shows that combined treatment with Fenofibrate plus Benznidazole is able both to reverse the cardiac dysfunction associated with the ongoing inflammatory response and fibrosis and to attain parasite clearance in an experimental model of Chagas disease.

Macrophages derived from septic mice modulate nitric oxide synthase and angiogenic mediators in the heart.

Macrophages (Mps) can exert the defense against invading pathogens. During sepsis, bacterial lipopolisaccharide (LPS) activates the production of inflammatory mediators by Mps. Nitric oxide synthase (NOS) derived-nitric oxide (NO) is one of them. Besides, Mps may produce pro-angiogenic molecules such as vascular endothelial growth factor-A (VEGF-A) and metalloproteinases (MMPs). The mechanisms involved in the cardiac neovascular response by Mps during sepsis are not completely known. We investigated the ability of LPS-treated Mps from septic mice to modulate the behavior of cardiac cells as producers of NO and angiogenic molecules. In vivo LPS treatment (0.1 mg/mouse) increased NO production more than fourfold and induced de novo NOS2 expression in Mps. Immunoblotting assays also showed an induction in VEGF-A and MMP-9 expression in lysates obtained from LPS-treated Mps, and MMP-9 activity was detected by zymography in cell supernatants. LPS-activated Mps co-cultured with normal heart induced the expression of CD31 and VEGF-A in heart homogenates and increased MMP-9 activity in the supernatants. By immunohistochemistry, we detected new blood vessel formation in hearts cultured with LPS treated Mps. When LPS-stimulated Mps were co-cultured with isolated cardiomyocytes in a transwell assay, the expression of NOS2, VEGF-A and MMP-9 was induced in cardiac cells. In addition, MMP-9 activity was up-regulated in the supernatant of cardiomyocytes. The latter was due to NOS2 induction in Mps from in vivo LPS-treated mice. In conclusion LPS-treated Mps are inducers of inflammatory/angiogenic mediators in cardiac cells, which could be triggering neovascularization, as an attempt to improve cardiac performance in sepsis.