in-vitro-differentiation-human-dendritic-cells-their-markers

<em>In Vitro</em> Differentiation of Human Dendritic Cells and their Markers in <em>Leishmania</em> Infection

Leishmaniasis comprises a collection of clinical manifestations associated with the infection of obligate intracellular protozoans, Leishmania. The life ...

Gonçalo Moniz Institute

CBA mouse macrophages control  infection yet are permissive to . Few studies have been conducted to assess the role played by autophagy in  infection. Therefore, we assessed whether the autophagic response of infected macrophages may account for the differential behavior of these two parasite strains. After 24 h of infection, the LC3-II/Act ratio increased in both - and -infected macrophages compared to uninfected controls, but less than in chloroquine-treated cells. This suggests that  and  activate autophagy in infected macrophages, without altering the autophagic flux. Furthermore, infected cells exhibited higher percentages of DQ-BSA-labeled parasitophorous vacuoles (50%) than those infected by  (25%). However, - and -induced parasitophorous vacuoles accumulated LysoTracker similarly, indicating that the acidity in both compartment was equivalent. At as early as 30 min, endogenous LC3 was recruited to both - and -induced parasitophorous vacuoles, while after 24 h a greater percentage of LC3 positive vacuoles was observed in -infected cells (42.36%) compared to those infected by  (18.10%). Noteworthy, principal component analysis (PCA) and an hierarchical cluster analysis completely discriminated -infected macrophages from -infected cells accordingly to infection intensity and autophagic features of parasite-induced vacuoles. Then, we evaluated whether the modulation of autophagy exerted an influence on parasite infection in macrophages. No significant changes were observed in both infection rate or parasite load in macrophages treated with the autophagic inhibitors wortmannin, chloroquine or VPS34-IN1, as well as with the autophagic inducers rapamycin or physiological starvation, in comparison to untreated control cells. Interestingly, both autophagic inducers enhanced intracellular  and  viability, while the pharmacological inhibition of autophagy exerted no effects on intracellular parasite viability. We also demonstrated that autophagy induction reduced NO production by - and -infected macrophages but not alters arginase activity. These findings provide evidence that although -induced parasitophorous vacuoles recruit LC3 more markedly,  and  similarly activate the autophagic pathway in CBA macrophages. Interestingly, the exogenous induction of autophagy favors  intracellular viability to a greater extent than  related to a reduction in the levels of NO.

Autophagic Induction Greatly Enhances  Intracellular Survival Compared to  in CBA/j-Infected Macrophages.

The current long-term treatment for leishmaniasis causes severe side effects and resistance in some cases. An evaluation of the anti-leishmanial potential of an HSP90-inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), demonstrated its potent effect against  spp.  and . We have previously shown that 17-AAG can kill  promastigotes with an IC of 65 nM and intracellular amastigote at concentrations as low as 125 nM. As this compound presents low solubility and high toxicity in human clinical trials, we prepared an inclusion complex containing hydroxypropyl-β-cyclodextrin and 17-AAG (17-AAG:HPβCD) to improve its solubility. This complex was characterized by scanning electron microscopy, and X-ray diffraction. Liposomes-containing 17-AAG:HPβCD was prepared and evaluated for encapsulation efficiency (EE%), particle size, polydispersity index (PDI), pH, and zeta potential, before and after accelerated and long-term stability testing. An evaluation of leishmanicidal activity against promastigotes and intracellular amastigotes of  was also performed. The characterization techniques utilized confirmed the formation of the inclusion complex, HPβCD:17-AAG, with a resulting 33-fold-enhancement in compound water solubility. Stability studies revealed that 17-AAG:HPβCD-loaded liposomes were smaller than 200 nm, with 99% EE. Stability testing detected no alterations in PDI that was 0.295, pH 7.63, and zeta potential +22.6, suggesting liposome stability, and suitability for evaluating leishmanicidal activity. Treatment of infected macrophages with 0.006 nM of 17-AAG:HPβCD or 17-AAG:HPβCD-loaded liposomes resulted in almost complete amastigote clearance inside macrophages after 48 h. This reduction is similar to the one observed in infected macrophages treated with 2 μM amphotericin B. Our results showed that nanotechnology and drug delivery systems could be used to increase the antileishmanial efficacy and potency of 17-AAG , while also resulting in reduced toxicity that indicates these formulations may represent a potential therapeutic strategy against leishmaniasis.

Encapsulation of the HSP-90 Chaperone Inhibitor 17-AAG in Stable Liposome Allow Increasing the Therapeutic Index as Assessed, , on  Amastigotes-Hosted in Mouse CBA Macrophages.

Leishmaniasis is a neglected disease that affects millions of individuals around the world. Regardless of clinical form, treatment is based primarily on the use of pentavalent antimonials. However, such treatments are prolonged and present intense side effects, which lead to patient abandonment in many cases. The search for chemotherapeutic alternatives has become a priority. Heat Shock Protein 90 (Hsp90) inhibitors have recently come under investigation due to antiparasitic activity in Plasmodium sp., Trypanosoma sp. and Leishmania sp. Some of these inhibitors, such as geldanamycin and its analogs, 17-AAG and 17-DMAG, bind directly to Hsp90, thereby inhibiting its activity. Previous studies have demonstrated that different parasite species are more susceptible to some of these inhibitors than host cells. We hypothesized that this increased susceptibility may be due to differences in binding of Hsp90 inhibitors to Leishmania protein compared to host protein. Based on the results of the in silico approach used in the present study, we propose that geldanamycin, 17-AAG and 17-DMAG present an increased tendency to bind to the N-terminal domain of Leishmania amazonensis Hsp83 in comparison to human Hsp90. This could be partially explained by differences in intermolecular interactions between each of these inhibitors and Hsp83 or Hsp90. The present findings demonstrate potential for the use of these inhibitors in the context of anti-Leishmania therapy.

A docking-based structural analysis of geldanamycin-derived inhibitor binding to human or Leishmania Hsp90.

Leishmaniasis is a spectrum of neglected tropical diseases and its cutaneous form (CL) is characterized by papillary or ulcerated skin lesions that negatively impact patients' quality of life. Current CL treatments suffer limitations, such as severe side effects and high cost, making the search for new therapeutic alternatives an imperative. In this context, heat shock protein 90 (Hsp90) could present a novel therapeutic target, as evidence suggests that Hsp90 inhibitors, such as 17-Dimethylaminoethylamino-17-Demethoxygeldanamycin (17-DMAG), may represent promising chemotherapeutic agents against CL. As innovative input for formulation development of 17-DMAG, nano-based drug delivery systems could provide controlled release, targeting properties, and reduced drug toxicity. In this work, a double emulsion method was used to develop poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing 17-DMAG. The nanoparticle was developed using two distinct protocols: Protocol 1 (P1) and Protocol 2 (P2), which differed concerning the organic solvent (acetone or dichloromethane, respectively) and procedure used to form double-emulsions (Ultra-Turrax® homogenization or sonication, respectively). The nanoparticles produced by P2 were comparatively smaller (305.5 vs. 489.0 nm) and more homogeneous polydispersion index (PdI) (0.129 vs. 0.33) than the ones made by P1. Afterward, the P2 was optimized and the best composition consisted of 2 mg of 17-DMAG, 100 mg of PLGA, 5% of polyethylene glycol (PEG 8000), 1.5 mL of the internal aqueous phase, 1% of polyvinyl alcohol (PVA), and 4 mL of the organic phase. Optimized P2 nanoparticles had a particle size of 297.2 nm (288.6-304.1) and encapsulation efficacy of 19.35% (15.42-42.18) by the supernatant method and 31.60% (19.9-48.79) by the filter/column method. Release kinetics performed at 37°C indicated that ~16% of the encapsulated 17-DMAG was released about to 72 h. In a separate set of experiments, a cell uptake assay employing confocal fluorescence microscopy revealed the internalization by macrophages of P2-optimized rhodamine B labeled nanoparticles at 30 min, 1, 2, 4, 6, 24, 48, and 72 h. Collectively, our results indicate the superior performance of P2 concerning the parameters used to assess nanoparticle development. Therefore, these findings warrant further research to evaluate optimized 17-DMAG-loaded nanoparticles (NP2-17-DMAG) for toxicity and antileishmanial effects  and .

Development and Characterization of PLGA Nanoparticles Containing 17-DMAG, an Hsp90 Inhibitor.

The heat shock protein 90 (Hsp90) is thought to be an excellent drug target against parasitic diseases. The leishmanicidal effect of an Hsp90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), was previously demonstrated in both in vitro and in vivo models of cutaneous leishmaniasis. Parasite death was shown to occur in association with severe ultrastructural alterations in , suggestive of autophagic activation. We hypothesized that 17-AAG treatment results in the abnormal activation of the autophagic pathway, leading to parasite death. To elucidate this process, experiments were performed using transgenic parasites with GFP-ATG8-labelled autophagosomes. Mutant parasites treated with 17-AAG exhibited autophagosomes that did not entrap cargo, such as glycosomes, or fuse with lysosomes. ATG5-knockout (Δ) parasites, which are incapable of forming autophagosomes, demonstrated lower sensitivity to 17-AAG-induced cell death when compared to wild-type (WT) , further supporting the role of autophagy in 17-AAG-induced cell death. In addition, Hsp90 inhibition resulted in greater accumulation of ubiquitylated proteins in both WT- and Δ-treated parasites compared to controls, in the absence of proteasome overload. In conjunction with previously described ultrastructural alterations, herein we present evidence that treatment with 17-AAG causes abnormal activation of the autophagic pathway, resulting in the formation of immature autophagosomes and, consequently, incidental parasite death.

17-AAG-Induced Activation of the Autophagic Pathway in  Is Associated with Parasite Death.

Editorial: Early Events During Host Cell-Pathogen Interaction.

, an intracellular parasite species, causes lesions on the skin and in the mucosa and internal organs. The dissemination of infected host cells containing  is crucial to parasite survival and the establishment of infection. Migratory phenomena and the mechanisms underlying the dissemination of -infected human dendritic cells (hDCs) remain poorly understood. The present study aimed to investigate differences among factors involved in hDC migration by comparing infection with visceral leishmaniasis (VL) induced by  with diverse clinical forms of tegumentary leishmaniasis (TL) induced by  or . Following the infection of hDCs by isolates obtained from patients with different clinical forms of , the formation of adhesion complexes, actin polymerization, and CCR7 expression were evaluated. We observed increased hDC migration following infection with isolates of  (VL), as well as disseminated (DL) and diffuse (DCL) forms of cutaneous leishmaniasis (CL) caused by  and , respectively. Increased expression of proteins involved in adhesion complex formation and actin polymerization, as well as higher CCR7 expression, were seen in hDCs infected with , DL and DCL isolates. Together, our results suggest that hDCs play an important role in the dissemination of  parasites in the vertebrate host.

Article	Year
Autophagic Induction Greatly Enhances Intracellular Survival Compared to in CBA/j-Infected Macrophages. Frontiers in microbiology\| PubMed ID: 30158914	2018
Encapsulation of the HSP-90 Chaperone Inhibitor 17-AAG in Stable Liposome Allow Increasing the Therapeutic Index as Assessed, , on Amastigotes-Hosted in Mouse CBA Macrophages. Frontiers in cellular and infection microbiology\| PubMed ID: 30214897	2018
A docking-based structural analysis of geldanamycin-derived inhibitor binding to human or Leishmania Hsp90. Scientific reports\| PubMed ID: 31611575	2019
Development and Characterization of PLGA Nanoparticles Containing 17-DMAG, an Hsp90 Inhibitor. Frontiers in chemistry\| PubMed ID: 34055735	2021
17-AAG-Induced Activation of the Autophagic Pathway in Is Associated with Parasite Death. Microorganisms\| PubMed ID: 34069389	2021
Editorial: Early Events During Host Cell-Pathogen Interaction. Frontiers in cellular and infection microbiology\| PubMed ID: 34095001	2021
-Induced Dendritic Cell Migration and Its Potential Contribution to Parasite Dissemination. Microorganisms\| PubMed ID: 34207943	2021

Patrícia S. T. Veras

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