Dynamics of the Trypanosoma cruzi infection in adipose tissue: Assessing gene expression of PNPLA2, FASN, and ACAT1 under Benzonidazole treatment and indirect mononuclear immune cells interaction.

Trypanosoma cruzi is a parasite with a high capacity to adapt to the host. Animal models have already demonstrated that the tropism of this parasite occurs not only in cardiac/digestive tissues but also in adipose tissue (AT). That said, the consequences ofT. cruziinfection for AT and the implications of treatment with Benzonidazole in this tissue are under discussion. Here, we tested the hypothesis that T. cruzi infection in adipose tissue upon treatment with Benzonidazole (Bz) and the interaction of mononuclear immune cells (PBMC) influences the relative expression of ACAT1, FASN, and PNPLA2 genes. Thus, stem cells derived from adipose tissue (ADSC) after adipogenic differentiation were indirectly cultivated with PBMC after infection with the T. cruzi Y strain and treatment with Bz. We use the TcSAT-IAM system and RT-qPCR to evaluate the parasite load and the relative quantification (ΔCt) of the ACAT1, FASN, and PNPLA2 genes. Our results demonstrate that treatment with Bz did not reduce adipocyte infection in the presence (p-value: 0.5796) or absence (p-value: 0.1854) of cultivation with PBMC. In addition, even though there is no statistical difference when compared to the control group (AT), T. cruzi induces the FASN expression (Rq: 14.00). However, treatment with Bz in AT suggests the increases of PNPLA2 expression levels (Rq: 12.58), even in the absence of T. cruzi infection. During indirect cultivation with PBMC, T. cruzi smooths the expression of PNPLA2 (Rq: 0.824) and instigates the expression of ACAT1 (Rq: 1.632) and FASN (Rq: 1.394). Furthermore, the treatment with Bz during infection induces PNPLA2 expression (Rq: 1.871), maintaining FASN expression levels (Rq: 1.334). Given this, our results indicate that treatment with Benzonidazole did not decrease T. cruzi infection in adipose tissue. However, treating the adipocyte cells with Bz during the interaction with PBMC cells influences the lipid pathways scenario, inducing lipolytic metabolism through the expression of PNPLA2.

Interaction between peripheral blood mononuclear cells and Trypanosoma cruzi-infected adipocytes: implications for treatment failure and induction of immunomodulatory mechanisms in adipose tissue.

Background/Introduction: Adipose tissue (AT) has been highlighted as a promising reservoir of infection for viruses, bacteria and parasites. Among them is Trypanosoma cruzi, which causes Chagas disease. The recommended treatment for the disease in Brazil is Benznidazole (BZ). However, its efficacy may vary according to the stage of the disease, geographical origin, age, immune background of the host and sensitivity of the strains to the drug. In this context, AT may act as an ally for the parasite survival and persistence in the host and a barrier for BZ action. Therefore, we investigated the immunomodulation of T. cruzi-infected human AT in the presence of peripheral blood mononuclear cells (PBMC) where BZ treatment was added. Methods: We performed indirect cultivation between T. cruzi-infected adipocytes, PBMC and the addition of BZ. After 72h of treatment, the supernatant was collected for cytokine, chemokine and adipokine assay. Infected adipocytes were removed to quantify T. cruzi DNA, and PBMC were removed for immunophenotyping. Results: Our findings showed elevated secretion of interleukin (IL)-6, IL-2 and monocyte chemoattractant protein-1 (MCP-1/CCL2) in the AT+PBMC condition compared to the other controls. In contrast, there was a decrease in tumor necrosis factor (TNF) and IL-8/CXCL-8 in the groups with AT. We also found high adipsin secretion in PBMC+AT+T compared to the treated condition (PBMC+AT+T+BZ). Likewise, the expression of CD80+ and HLA-DR+ in CD14+ cells decreased in the presence of T. cruzi. Discussion: Thus, our findings indicate that AT promotes up-regulation of inflammatory products such as IL-6, IL-2, and MCP-1/CCL2. However, adipogenic inducers may have triggered the downregulation of TNF and IL-8/CXCL8 through the peroxisome proliferator agonist gamma (PPAR-g) or receptor expression. On the other hand, the administration of BZ only managed to reduce inflammation in the microenvironment by decreasing adipsin in the infected culture conditions. Therefore, given the findings, we can see that AT is an ally of the parasite in evading the host's immune response and the pharmacological action of BZ.

TNF blockers alone and associated with Benznidazole impact in vitro cytokine dynamics in chronic Chagas disease.

Studies involving the immune response in Chagas disease suggest an imbalance in the immune response of symptomatic patients, with an inflammatory profile dominating in Chagas heart disease, mainly by tumour necrosis factor (TNF). TNF is considered a key cytokine in immunopathology in chronic carriers in several processes during the immune response. Our work aimed to evaluate regulatory (interleukin [IL]-4 and IL-10) and inflammatory (TNF, interferon-gamma [IFN-γ], IL-2 and IL-6) cytokines in peripheral blood mononuclear cells culture supernatants. of affected patients with undetermined clinical forms-IND (n = 13) mild heart form-CARD1 (n = 13) and severe cardiac form-CARD2 (n = 16), treated in vitro with two TNF blockers, Adalimumab (ADA) and Etanercept (ETA) alone or in association with Benznidazole (BZ). The results indicate that ADA was more competent in blocking TNF (compared to ETA) in all groups but with much lower levels in the CARD2 group. ETA statistically decreased TNF levels only in the CARD2 group. IFN-γ increased in the CARD2 group after treatment with ETA relative to ADA. IL-4 had its levels decreased when treated by both drugs. IL-2 was detected in cells from CARD2 carriers compared to the NEG group after treatment with both drugs. The association with BZ decreased levels of IL-2/TNF and increased IL-4. These data reinforce the participation of TNF in severe Chagas heart disease and bring perspectives on using these blockers in the immunological treatment of Chagas disease since the use of BZ is extremely limited in these patients.

Benznidazole treatment decreases IL-6 levels in Trypanosoma cruzi-infected human adipocytes differentiated from adipose tissue-derived stem cells.

BACKGROUND: Trypanosoma cruzi, which causes Chagas disease (CD), is a versatile haemoparasite that uses several strategies to evade the host's immune response, including adipose tissue (AT), used as a reservoir of infection. As it is an effective barrier to parasite evasion, the effectiveness of the drug recommended for treating CD, Benznidazole (BZ), may be questionable. OBJECTIVE: To this end, we evaluated the parasite load and immunomodulation caused by BZ treatment in the culture of adipocytes differentiated from human adipose tissue-derived stem cells (ADSC) infected with T. cruzi. METHODS: The ADSC were subjected to adipogenic differentiation. We then carried out four cultures in which we infected the differentiated AT with trypomastigote forms of the Y strain of T. cruzi and treated them with BZ. After the incubation, the infected AT was subjected to quantitative polymerase chain reaction (qPCR) to quantify the parasite load and transmission electron microscopy (TEM) to verify the infection. The supernatant was collected to measure cytokines, chemokines, and adipokines. FINDINGS: We found elevated secretion of IL-6, CXCL-10/IP-10, CCL2/MCP-1, CCL5/RANTES, and leptin in infected fat cells. However, treatment with BZ promoted a decrease in IL-6. MAIN CONCLUSION: Therefore, we believe that BZ has a beneficial role as it reduces inflammation in infected fat cells.

Outbreak of Chagas disease in Brazil: Validation of a molecular diagnostic method.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi (T. cruzi), affects millions of people worldwide. Polymerase Chain Reaction (PCR) and real-time quantitative PCR (qPCR) have been used as tools to monitor parasitic levels in the bloodstream of individuals exposed to infection, thus enabling the monitoring of relapses and the effectiveness of therapy, for example. The aim of this study was to evaluate the TcSAT-IAM system, developed by our research group, on samples from patients with suspected Chagas disease infection. Initially, primer systems were developed for the detection of the nuclear DNA (SAT-DNA) from T. cruzi (TcSAT-IAM). The Cruzi system, predicted in the literature, and TcSAT-IAM were then evaluated in relation to their analytical sensitivity, specificity and efficiency. Afterwards, the applicability of the qPCR technique using both systems (separately) for the diagnosis of acute CD was evaluated in samples from 77 individuals exposed to the outbreak that occurred in Pernambuco-Brazil, relating the results obtained to those of the classical diagnostic methods recommended for this stage of the infection. TcSAT-IAM and Cruzi had a detection limit of 1 fg of target DNA (0,003 parasites). Thirty-eight cases were recorded, 28 by laboratory criteria and 10 by clinical and epidemiological criteria. Blood samples from 77 subjects were submitted to qPCR by both systems, reaching an agreement of 89.61% between them. After analyzes between systems and diagnostic criteria, the TcSAT-IAM showed sensitivity and specificity of 52.36% (CI 37.26-67.52) and 92.31% (CI 79.68-97.35), respectively, accuracy of 72.73% and moderate agreement. The TcSAT-IAM showed an accuracy of 72.58% and 75% in relation to parasitological and serological tests (IgM anti-T. cruzi), respectively. Therefore, quantitative PCR should be incorporated into the diagnosis of suspected acute cases of Chagas disease.

Green microalgae as a potential source of trypanocide compounds.

Due to the limitations of Chagas disease therapy, microalgae can be promising in the search of new trypanocidal compounds, since these organisms produce bioactive compounds with large pharmaceutical applications, including antiparasitic effects. In this work, trypanocidal activity of aqueous extract of Tetradesmus obliquus and, for the first time, aqueous extract of Chlorella vulgaris, were evaluated against trypomastigote forms of Trypanosoma cruzi. In addition, cytotoxic activity in Vero cells was evaluated. Our results showed that C. vulgaris and T. obliquus present trypanocidal activity (IC50 = 32.9 µg ml-1 and 36.4 µg ml-1, respectively), however, C. vulgaris did not present cytotoxic effects in Vero cells (CC50 > 600 µg ml-1) and displayed a higher selectivity against trypomastigotes forms of T. cruzi (SI > 18). Thus, microalgae extracts, such as aqueous extract of C. vulgaris, are promising potential candidates for the development of natural antichagasic drugs.

Benznidazole treatment decreases IL-6 levels in Trypanosoma cruzi-infected human adipocytes differentiated from adipose tissue-derived stem cells

BACKGROUND Trypanosoma cruzi, which causes Chagas disease (CD), is a versatile haemoparasite that uses several strategies to evade the host's immune response, including adipose tissue (AT), used as a reservoir of infection. As it is an effective barrier to parasite evasion, the effectiveness of the drug recommended for treating CD, Benznidazole (BZ), may be questionable. OBJECTIVE To this end, we evaluated the parasite load and immunomodulation caused by BZ treatment in the culture of adipocytes differentiated from human adipose tissue-derived stem cells (ADSC) infected with T. cruzi. METHODS The ADSC were subjected to adipogenic differentiation. We then carried out four cultures in which we infected the differentiated AT with trypomastigote forms of the Y strain of T. cruzi and treated them with BZ. After the incubation, the infected AT was subjected to quantitative polymerase chain reaction (qPCR) to quantify the parasite load and transmission electron microscopy (TEM) to verify the infection. The supernatant was collected to measure cytokines, chemokines, and adipokines. FINDINGS We found elevated secretion of IL-6, CXCL-10/IP-10, CCL2/MCP-1, CCL5/RANTES, and leptin in infected fat cells. However, treatment with BZ promoted a decrease in IL-6. MAIN CONCLUSION Therefore, we believe that BZ has a beneficial role as it reduces inflammation in infected fat cells.

Is a negative correlation between sTNFR1 and TNF in patients with chronic Chagas disease the key to clinical progression?

Soluble TNF receptors (sTNFR1 and sTNFR2) are natural endogenous inhibitors of TNF and are elevated in inflammatory, autoimmune, and chronic degenerative diseases. In Chagas disease, pleiotropic cytokine TNF is considered key in immunopathology. Thus, we aimed to evaluate the levels of TNF, sTNFR1, and sTNFR2 in the serum of patients with chronic Chagas disease. TNF and its soluble receptors were quantified using Cytometric Bead Array in the serum of 132 patients, of which 51 had the indeterminate form (IND), 39 the mild cardiac form (CARD 1), 42 the severe cardiac form (CARD 2), and 20 non-infected individuals (NI). The results indicate that the soluble receptors may regulate TNF in Chagas disease, as their leves were higher in T. cruzi-infected individuals when compared to non-infected individuals. We found a moderate negative correlation between sTNFR1 and TNF in individuals with the IND form, suggesting a relationship with non-progression to more severe forms, such as heart disease. sTNFR1 and sTNFR2 were increased in all clinical forms, but with a moderate positive correlation in more severe patients (r = 0.50 and p = 0.0005). TNF levels showed no statistical differences in the groups of patients. These findings suggest the importance of the endogenous balance of the levels of soluble TNF receptors in the protection and balance in patients with chronic Chagas disease, besides revealing the immunological complexity in chronic T. cruzi-infected individuals.

A Trypsin Inhibitor from Moringa oleifera Flowers Modulates the Immune Response In Vitro of Trypanosoma cruzi-Infected Human Cells.

Trypanosoma cruzi causes the lethal Chagas disease, which is endemic in Latin America. Flowers of Moringa oleifera (Moringaceae) express a trypsin inhibitor (MoFTI) whose toxicity to T. cruzi trypomastigotes was previously reported. Here, we studied the effects of MoFTI on the viability of human peripheral blood mononuclear cells (PBMCs) as well as on the production of cytokines and nitric oxide (NO) by T. cruzi-infected PBMCs. Incubation with MoFTI (trypsin inhibitory activity: 62 U/mg) led to lysis of trypomastigotes (LC50 of 43.5 µg/mL) but did not affect the viability of PBMCs when tested at concentrations up to 500 µg/mL. A selectivity index > 11.48 was determined. When T. cruzi-infected PBMCs were treated with MoFTI (43.5 or 87.0 µg/mL), the release of the pro-inflammatory cytokine TNF-α and INF-γ, as well as of NO, was stimulated. The release of the anti-inflammatory cytokine IL-10 also increased. In conclusion, the toxicity to T. cruzi and the production of IL-10 by infected PBMCs treated with MoFTI suggest that this molecule may be able to control parasitemia while regulating the inflammation, preventing the progress of Chagas disease. The data reported here stimulate future investigations concerning the in vivo effects of MoFTI on immune response in Chagas disease.

Evaluation of cytotoxic, immunomodulatory and antibacterial activities of aqueous extract from leaves of Conocarpus erectus Linnaeus (Combretaceae).

OBJECTIVES: This work evaluated the antibacterial activity, cytotoxicity and immunomodulatory effect on human peripheral blood mononuclear cells (PBMCs) promoted by aqueous extract from Conocarpus erectus leaves (AELCe). METHODS: The extract was characterized by thin layer chromatography and ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). Cytotoxicity of AELCe (6.25-50 µg/ml) was investigated using annexin V and propidium iodide. Cytokine and nitric oxide levels in PBMCs culture supernatants exposed or not to AELCe (12.5 µg/ml) were determined, and antibacterial activity was evaluated by disc diffusion and broth microdilution methods. KEY FINDINGS: AELCe contained 3',4'-OH flavonoids, phenylpropanoglycosides, saponins, polymeric proanthocyanidins and hydrolysable tannins. Moreover, 10 other compounds were identified through UPLC-MS technique. AELCe did not affect lymphocyte viability at 6.25 and 12.5 µg/ml. IL-2, IL-10, TNF-α, IFN-γ and nitric oxide was produced in higher levels by cells treated with AELCe. Proliferation and activation of CD8+ T lymphocytes were also stimulated. AELCe showed bacteriostatic activity against clinical and antibiotic-resistant strains of Staphylococcus aureus (MIC between 250 and 1000 µg/ml). CONCLUSIONS: AELCe showed a moderate bacteriostatic activity and promoted an immunomodulatory status through higher production of Th1 cytokines, nitric oxide release and T CD8+ lymphocytes stimulation.

Lectin from inflorescences of ornamental crop Alpinia purpurata acts on immune cells to promote Th1 and Th17 responses, nitric oxide release, and lymphocyte activation.

Alpinia purpurata is an ornamental crop known as a source of bioactive molecules. This is the first study to report isolation of a lectin (carbohydrate-binding protein) from A. purpurata inflorescences (ApuL). The immunomodulatory potential of ApuL was evaluated by investigating its effects on the production of cytokines and release of nitric oxide by human peripheral blood mononuclear cells (PBMCs). In addition, the differentiation and activation of lymphocytes treated with ApuL was evaluated by immunophenotyping assays. ApuL is an acidic and oligomeric protein with native molecular mass of 34kDa. The hemagglutinating activity (HA) of ApuL was inhibited by the glycoproteins fetuin and ovalbumin, was resistant to heating at 100°C and stimulated in the presence of calcium and magnesium ions. ApuL showed highest HA at pH 7.5 but failed to agglutinate erythrocytes at pH 8.0 and 9.0. ApuL induced the release of cytokines belonging to Th1 (IFN-γ, TNF-α, and IL-6) and Th17 (IL-17A) profiles as well as of nitric oxide, stimulating a pro-inflammatory environment. Moreover, ApuL also stimulated the production of IL-10, an anti-inflammatory cytokine with regulatory role. Incubation with lectin resulted in differentiation and activation of both T CD8+ and CD4+ subsets of lymphocytes, as evident from the expression of the CD28 costimulatory molecule. In conclusion, A. purpurata inflorescence is a source of an immunomodulatory lectin with potential immunoregulatory application, thereby adding biotechnological value to this ornamental crop.

Microgramma vacciniifolia (Polypodiaceae) fronds contain a multifunctional lectin with immunomodulatory properties on human cells.

In this study, we report the purification and characterization of a multifunctional lectin (MvFL) from Microgramma vacciniifolia fronds as well as its immunomodulatory properties on human peripheral blood mononuclear cells (PBMCs). MvFL (pI 4.51; 54kDa) is a glycoprotein able to inhibit trypsin activity and that has sequence similarities (32% coverage) with a plant RNA-binding protein. Hemagglutinating activity of MvFL was not altered by heating at 100°C for 30min, but was reduced in alkaline pH (8.0 and 9.0). Fluorimetric analyses showed that this lectin did not undergo marked conformational changes when heated. However, the MvFL conformation changed depending on the pH. MvFL at 6.25-25µg/mL was not cytotoxic to lymphocytes present among PBMCs. The PBMCs incubated for 24h with the lectin (12.5µg/mL) showed increased TNF-α, IFN-γ, IL-6, IL-10, and nitric oxide production. MvFL also stimulated T lymphocytes from PBMCs to differentiate into CD8+ cells. The activation (indicated by CD28 expression) of these cells was also stimulated. In conclusion, MvFL is a heat-stable and multifunctional protein, with both lectin and trypsin inhibitor activities, and capable of inducing predominantly a Th1 response in human PBMCs as well as activation and differentiation of T lymphocytes.