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.

Benzonidazole treatment has a beneficial effect on cells infected with the Colombian strain of Trypanosoma cruzi.

Benznidazole (Bz) is the recommended drug for the treatment of Chagas disease; however, its efficacy may vary according to the sensitivity of Trypanosoma cruzi strains to the drug and host immune background. The study evaluated the immune response of peripheral blood mononuclear cells (PBMC) that were infected in vitro with the Colombian strain (Col) and treated with Bz. The co-cultures were incubated for 24 h, 5 and 10 days, where cytokine dosage was performed in the supernatant and evaluation of the cells for CD28+ and CTLA-4+ molecules in CD4+ and CD8+ lymphocytes, and CD80+ , CD86+ and HLA-DR+ in CD14+ cells. The results showed that Col induced a strong inflammatory response, with an increase in IFN-γ and TNF early in the infection (24 h), however, from 5 days of infection on, TNF production declined, and IL-10 production increased, which may be associated with a control mechanism of the exacerbated inflammatory response. The Bz treatment did not significantly alter the frequencies of the phenotypes evaluated both T cell subsets and CD14+ cells. Therefore, this study reinforces the need for typing the patient's strain to guide therapy and promote individualized treatment protocols due to the heterogeneous genetic background among T. cruzi strains.

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.

Effects of Migonemyia migonei salivary gland homogenates on Leishmania (Viannia) braziliensis infection in BALB/c mice.

Cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis is the most widespread clinical form of leishmaniasis in the Americas. Migonemyia migonei is a widely distributed phlebotomine sand fly species in Brazil and has been implicated as a vector for L. (V.) braziliensis. In the present study, we investigated the effects of salivary gland homogenates (SGH) of Mg. migonei on the course of L. (V.) braziliensis infection in BALB/c mice. Mice were separated into four groups (six mice per group): CTRL (uninfected mice); SGH (mice inoculated with Mg. migonei SGH); SGH+LEISH (mice inoculated with Mg. migonei SGH plus L. (V.) braziliensis promastigotes); LEISH (mice inoculated with L. (V.) braziliensis promastigotes). Mice were followed up for 8 weeks and the cellular immune response was evaluated by flow cytometry at the end of the experiment. Analysis of cytokine production by splenic cells stimulated with 0.5 SGH, 0.25 SGH of Mg. migonei or L. (V.) braziliensis soluble antigen stimulation (LSA) demonstrated that upon stimulation with SGH 0.25, the production of IL-17A and TNF was not sustained in the SGH group, with decreasing levels of these cytokines after 5 days compared to 3 days of incubation. Analyzing the production of cytokines after LSA stimulation, we observed lower levels of IL-17A in the SGH group after 5 days compared to 3 days. The same was observed for IFN-γ in the SGH group. Yet, the levels of TNF were significantly higher in the LEISH group after 5 days compared to 3 days. Among SGH+LEISH and LEISH mice, three animals in each group developed skin lesions on the tail, the mean lesion size was significantly higher in the LEISH group. Our study suggests that Mg. migonei SGH may modulate BALB/c immune response, as reflected by the low production or early decrease of pro-inflammatory cytokines in splenic cell cultures following stimulation with L. (V.) braziliensis antigen. Our data also suggest that Mg. migonei saliva may reduce the lesion size in BALB/c mice, but further research with a larger sample size is needed to confirm this hypothesis.

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.

Antitumor activity of Moringa oleifera (drumstick tree) flower trypsin inhibitor (MoFTI) in sarcoma 180-bearing mice.

The plant Moringa oleifera is used as food and medicine. M. oleifera flowers are source of protein, fiber, and antioxidants, and are used to treat inflammation and tumors. This work evaluated the antitumor activity of the M. oleifera flower trypsin inhibitor (MoFTI) in sarcoma 180-bearing mice. Swiss female mice were inoculated with sarcoma 180 cells. Seven days later, the animals were treated intraperitoneally for 1 week with daily doses of PBS (control) or MoFTI (15 or 30 mg/kg). For toxicity assessment, water and food consumption, body and organ weights, histological alterations, and blood hematological and biochemical parameters were measured. Treatment with MoFTI caused pronounced reduction (90.1%-97.9%) in tumor weight. The tumors of treated animals had a reduced number of secondary vessels and lower gauge of the primary vessels compared to the control. No significant changes were observed in water and food consumption or in body and organ weights. Histopathological analysis did not indicate damage to the liver, kidneys, and spleen. In conclusion, MoFTI showed antitumor potential, with no clear evidence of toxicity.