The Paradoxical Leishmanicidal Effects of Superoxide Dismutase (SOD)-Mimetic Tempol in Leishmania braziliensis Infection in vitro.
ABSTRACT: Leishmaniasis is an infectious disease caused by protozoans of the genus Leishmania. The macrophage is the resident cell in which the parasite replicates and it is important to identify new compounds that can aid in parasite elimination since the drugs used to treat leishmaniasis are toxic and present side effects. We have previously shown that treatment of Leishmania braziliensis-infected macrophages with DETC (Diethyldithiocarbamate) induces parasite killing, in vivo. Thus, the objective of this study was to further evaluate the effect of oxidants and antioxidants in L. braziliensis-infected macrophages, following treatment with either oxidizing Hydrogen Peroxide, Menadione, DETC, or antioxidant [NAC (N-Acetyl-Cyteine), Apocynin, and Tempol] compounds. We determined the percentage of infected macrophages and number of amastigotes. Promastigote survival was also evaluated. Both DETC (SOD-inhibitor) and Tempol (SOD-mimetic) decreased the percentage of infected cells and parasite load. Hydrogen peroxide did not interfere with parasite burden, while superoxide-generator Menadione had a reducing effect. On the other hand, NAC (GSH-replenisher) and Apocynin (NADPH-oxidase inhibitor) increased parasite burden. Tempol surfaces as an interesting candidate for the chemotherapy of CL with an IC50 of 0.66 ± 0.08 mM and selectivity index of 151. While it remains obscure how a SOD-mimetic may induce leishmanicidal effects, we suggest the possibility of developing Tempol-based topical applications for the treatment of cutaneous leishmaniasis caused by L. braziliensis.
Project description:The treatment of leishmaniasis still relies on drugs with potentially serious adverse effects. Herein, we tested a topical formulation of bacterial cellulose (BC) membranes containing Diethyldithiocarbamate (DETC), a superoxide dismutase 1 inhibitor. Leishmania-infected macrophages exposed to BC-DETC resulted in parasite killing, without pronounced toxic effects to host cells. This outcome was associated with lower SOD1 activity and higher production of superoxide and cytokine mediators. Topical application of BC-DETC significantly decreased lesion size, parasite load and the inflammatory response at the infection site, as well as the production of both IFN-? and TNF. Combination of topical BC-DETC plus intraperitoneal Sbv also significantly reduced disease development and parasite load. The leishmanicidal effect of BC-DETC was extended to human macrophages infected with L. braziliensis, highlighting the feasibility of BC-DETC as a topical formulation for chemotherapy of cutaneous leishmaniasis caused by L. braziliensis.
Project description:The redox-sensitive Sp family transcription factor has been linked to the regulation of angiotensin II type 1 receptor (AT1R). However, the exact mechanism of AT1R regulation in renal cells is poorly understood. We tested the specificity of reactive oxygen species (ROS), superoxide vs. hydrogen peroxide (H2O2), and the specific role of Sp3 transcription factor, if any, in the regulation of AT1R in human kidney cells (HK2 cells). Superoxide dismutase (SOD) inhibitor diethyldithiocarbamate (DETC), but not H2O2 treatment, increased fluorescence levels of superoxide probe dihydroethidium (DHE). H2O2, but not DETC, treatment increased the fluorescence of the H2O2-sensitive probe dichloro-dihydro-fluorescein (DCFH). These data suggest that SOD inhibition by DETC increases the superoxide but not H2O2 and exogenously added H2O2 is not converted to superoxide in renal cells. Furthermore, DETC, but not H2O2, treatment increased nuclear accumulation of Sp3, which was attenuated with the superoxide dismutase (SOD)-mimetic tempol. DETC treatment also increased AT1R mRNA and protein levels that were attenuated with tempol, whereas H2O2 did not have any effects on AT1R mRNA. Moreover, Sp3 overexpression increased, while Sp3 depletion by siRNA decreased, protein levels of AT1R. In addition, Sp3 siRNA in the presence of DETC decreased AT1R protein expression. Furthermore, DETC treatment increased the levels of cell surface AT1R as measured by biotinylation and immunofluorescence studies. Angiotensin II increased PKC activity in vehicle-treated cells that further increased in DETC-treated cells, which was attenuated by AT1R blocker candesartan and SOD-mimetic tempol. Taken together, our results suggest that superoxide, but not H2O2, via Sp3 up-regulates AT1R expression and function in the renal cells.
Project description:BACKGROUND: Leishmaniasis remains a worldwide public health problem. The limited therapeutic options, drug toxicity and reports of resistance, reinforce the need for the development of new treatment options. Previously, we showed that 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), a Heat Shock Protein 90 (HSP90)-specific inhibitor, reduces L. (L.) amazonensis infection in vitro. Herein, we expand the current knowledge on the leishmanicidal activity of 17-AAG against cutaneous leishmaniasis, employing an experimental model of infection with L. (V.) braziliensis. METHODOLOGY/PRINCIPAL FINDINGS: Exposure of axenic L. (V.) braziliensis promastigotes to 17-AAG resulted in direct dose-dependent parasite killing. These results were extended to L. (V.) braziliensis-infected macrophages, an effect that was dissociated from the production of nitric oxide (NO), superoxide (O(-2)) or inflammatory mediators such as TNF-?, IL-6 and MCP-1. The leishmanicidal effect was then demonstrated in vivo, employing BALB/c mice infected with L. braziliensis. In this model, 17-AAG treatment resulted in smaller skin lesions and parasite counts were also significantly reduced. Lastly, 17-AAG showed a similar effect to amphotericin B regarding the ability to reduce parasite viability. CONCLUSION/SIGNIFICANCE: 17-AAG effectively inhibited the growth of L. braziliensis, both in vitro and in vivo. Given the chronicity of L. (V.) braziliensis infection and its association with mucocutaneous leishmaniasis, 17-AAG can be envisaged as a new chemotherapeutic alternative for cutaneous Leishmaniasis.
Project description:Treatments based on antimonials to cutaneous leishmaniasis (CL) entail a range of toxic side effects. Propolis, a natural compound widely used in traditional medical applications, exhibits a range of biological effects, including activity against infectious agents. The aim of this study was to test the potential leishmanicidal effects of different propolis extracts against Leishmania (Viannia) braziliensis promastigotes and intracellular amastigotes in vitro. Stationary-phase L. (V) braziliensis promastigotes were incubated with medium alone or treated with dry, alcoholic, or glycolic propolis extract (10, 50, or 100 μg/mL) for 96 h. Our data showed that all extracts exhibited a dose-dependent effect on the viability of L. (V) braziliensis promastigotes, while controlling the parasite burden inside infected macrophages. Dry propolis extract significantly modified the inflammatory profile of murine macrophages by downmodulating TGF-β and IL-10 production, while upmodulating TNF-α. All three types of propolis extract were found to reduce nitric oxide and superoxide levels in activated L. braziliensis-infected macrophages. Altogether, our results showed that propolis extracts exhibited a leishmanicidal effect against both stages of L. (V) braziliensis. The low cell toxicity and efficient microbicidal effect of alcoholic or glycolic propolis extracts make them candidates to an additive treatment for cutaneous leishmaniasis.
Project description:<i>Leishmania braziliensis</i> infection causes skin ulcers, typically found in localized cutaneous leishmaniasis (LCL). This tissue pathology associates with different modalities of cell necrosis, which are subverted by the parasite as a survival strategy. Herein we examined the participation of necroptosis, a specific form of programmed necrosis, in LCL lesions and found reduced RIPK3 and PGAM5 gene expression compared to normal skin. Assays using infected macrophages demonstrated that the parasite deactivates both RIPK3 and MLKL expression and that these molecules are important to control the intracellular <i>L. braziliensis</i> replication. Thus, LCL-related necroptosis may be targeted to control infection and disease immunopathology.
Project description:We show that increased plasma superoxide dismutase 1 (SOD1) levels are statistically significant predictors of the failure of pentavalent antimony treatment for cutaneous leishmaniasis caused by Leishmania braziliensis. In Leishmania amazonensis-infected patients, host SOD1 levels can be used to discriminate between localized and drug-resistant diffuse cutaneous leishmaniasis. Using in situ transcriptomics (nCounter), we demonstrate a significant positive correlation between host SOD1 and interferon ?/? messenger RNA (mRNA) levels, as well as interkingdom correlation between host SOD1 and parasite SOD2/4 mRNA levels. In human macrophages, in vitro treatment with SOD1 increases the parasite burden and induces a diffuse cutaneous leishmaniasis-like morphology. Thus, SOD1 is a clinically relevant biomarker and a therapeutic target in both localized and diffuse cutaneous leishmaniasis.
Project description:<i>Leishmania braziliensis</i> is an intracellular parasite that resides mostly in macrophages. Both the parasite genome and the clinical disease manifestations show considerable polymorphism. Clinical syndromes caused by <i>L. braziliensis</i> include localized cutaneous (CL), mucosal (ML), and disseminated leishmaniasis (DL). Our prior studies showed that genetically distinct <i>L. braziliensis</i> clades associate with different clinical types. Herein, we hypothesized that: (1) <i>L. braziliensis</i> induces changes in macrophage gene expression that facilitates infection; (2) infection of macrophages with strains associated with CL (clade B), ML (clade C), or DL (clade A) will differentially affect host cell gene expression, reflecting their different pathogenic mechanisms; and (3) differences between the strains will be reflected by differences in macrophage gene expression after initial exposure to the parasite. Human monocyte derived macrophages were infected with <i>L. braziliensis</i> isolates from clades A, B, or C. Patterns of gene expression were compared using Affymetrix DNA microarrays. Many transcripts were significantly decreased by infection with all isolates. The most dramatically decreased transcripts encoded proteins involved in signaling pathways, apoptosis, or mitochondrial oxidative phosphorylation. Some transcripts encoding stress response proteins were up-regulated. Differences between <i>L. braziliensis</i> clades were observed in the magnitude of change, rather than the identity of transcripts. Isolates from subjects with metastatic disease (ML and DL) induced a greater magnitude of change than isolates from CL. We conclude that <i>L. braziliensis</i> enhances its intracellular survival by inhibiting macrophage pathways leading to microbicidal activity. Parasite strains destined for dissemination may exert a more profound suppression than less invasive <i>L. braziliensis</i> strains that remain near the cutaneous site of inoculation.
Project description:Leishmania (subgenus Viannia) braziliensis is the causative agent of mucocutaneous leishmaniasis (ML) in South America, and ML is characterized by excessive T- and B-cell responses to the parasite. We speculate that the unbalanced production of inflammatory mediators in response to L. braziliensis infection contributes to cell recruitment and disease severity. To test this hypothesis, we first examined the response of peripheral blood mononuclear cells (PBMCs) from healthy volunteers to L. braziliensis infection. We observed that while L. braziliensis infection induced the production of chemokine (C-X-C motif) ligand 10 (CXCL10) and interleukin-10 (IL-10) in human PBMCs and macrophages (MPhis), enhanced expression of CXCL10 and its receptor, chemokine CXC receptor (CXCR3), was predominantly detected in CD14(+) monocytes. The chemoattractant factors secreted by L. braziliensis-infected cells were highly efficient in recruiting uninfected PBMCs (predominantly CD14(+) cells) through Transwell membranes. Serum samples from American tegumentary leishmaniasis (ATL) patients (especially the ML cases) had significantly higher levels of CXCL10, CCL4, and soluble tumor necrosis factor (TNF) receptor II (sTNFRII) than did those of control subjects. Our results suggest that, following L. braziliensis infection, the production of multiple inflammatory mediators by the host may contribute to disease severity by increasing cellular recruitment.
Project description:Host and parasite gene expression in skin biopsies from Leishmania braziliensis-infected patients were simultaneously analyzed using high throughput RNA-sequencing. Biopsies were taken from 8 patients with early cutaneous leishmaniasis and 17 patients with late cutaneous leishmaniasis. Although parasite DNA was found in all patient lesions at the time of biopsy, the patients could be stratified into two groups: one lacking detectable parasite transcripts (PTNeg) in lesions, and another in which parasite transcripts were readily detected (PTPos). These groups exhibited substantial differences in host responses to infection. PTPos biopsies contained an unexpected increase in B lymphocyte-specific and immunoglobulin transcripts in the lesions, and an upregulation of immune inhibitory molecules. Biopsies without detectable parasite transcripts showed decreased evidence for B cell activation, but increased expression of antimicrobial genes and genes encoding skin barrier functions. The composition and abundance of L. braziliensis transcripts in PTPos lesions were surprisingly conserved among all six patients, with minimal meaningful differences between lesions from patients with early and late cutaneous leishmaniasis. The most abundant parasite transcripts expressed in lesions were distinct from transcripts expressed in vitro in human macrophage cultures infected with L. amazonensis or L. major. Therefore in vitro gene expression in macrophage monolayers may not be a strong predictor of gene expression in lesions. Some of the most highly expressed in vivo transcripts encoded amastin-like proteins, hypothetical genes, putative parasite virulence factors, as well as histones and tubulin. In summary, RNA sequencing allowed us to simultaneously analyze human and L. braziliensis transcriptomes in lesions of infected patients, and identify unexpected differences in host immune responses which correlated with active transcription of parasite genes.
Project description:Infection with different Leishmania spp. protozoa can lead to a variety of clinical syndromes associated in many cases with inflammatory responses in the skin. Although macrophages harbor the majority of parasites throughout chronic infection, neutrophils are the first inflammatory cells to migrate to the site of infection. Whether neutrophils promote parasite clearance or exacerbate disease in murine models varies depending on the susceptible or resistant status of the host. Based on the hypothesis that neutrophils contribute to a systemic inflammatory state in humans with symptomatic L. braziliensis infection, we evaluated the phenotype of neutrophils from patients with cutaneous leishmaniasis (CL) during the course of L. braziliensis infection. After in vitro infection with L. braziliensis, CL patient neutrophils produced more reactive oxygen species (ROS) and higher levels of CXCL8 and CXCL9, chemokines associated with recruitment of neutrophils and Th1-type cells, than neutrophils from control healthy subjects (HS). Despite this, CL patient and HS neutrophils were equally capable of phagocytosis of L. braziliensis. There was no difference between the degree of activation of neutrophils from CL versus healthy subjects, assessed by CD66b and CD62L expression using flow cytometry. Of interest, these studies revealed that both parasite-infected and bystander neutrophils became activated during incubation with L. braziliensis. The enhanced ROS and chemokine production in neutrophils from CL patients reverted to baseline after treatment of disease. These data suggest that the circulating neutrophils during CL are not necessarily more microbicidal, but they have a more pro-inflammatory profile after parasite restimulation than neutrophils from healthy subjects.