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Objective: Visceral leishmaniasis is a neglected tropical disease that can be lethal if not treated. The available medicines have severe side effects, such as toxicity and drug resistance. Various investigations are looking into new anti-leishmanial compounds from natural products that have little impact on host cells. Lupeol, a triterpenoid present in the flora of many edible plants, has been shown to have antimicrobial properties. The present study investigated the immunomodulatory effects of lupeol on U937 macrophages infected with Leishmania donovani, focusing on the expression of key cytokines and enzymes involved in the immune response., Methods: U937 macrophages were infected with Leishmania donovani amastigotes and treated with varying concentrations of lupeol throughout three days. The expression levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10) were measured using real-time polymerase chain reaction (RT-PCR). A positive simulation of gene expression was estimated using ΔΔCT to assess relative expression., Results: The results demonstrated that lupeol significantly upregulated iNOS and TNF-α expression, especially at higher concentrations, indicating enhanced pro-inflammatory and anti-leishmanial activity. Interestingly, IL-10 expression also increased, suggesting a complex immunomodulatory role of lupeol that involves both pro-inflammatory and anti-inflammatory pathways. Pearson correlation analysis revealed a strong association between iNOS and TNF-α (0.97692), as well as a moderate correlation between iNOS and IL-10 (0.51603)., Conclusion: These findings suggest that lupeol may promote a balanced immune response, enhancing the body's ability to combat L. donovani while potentially mitigating excessive inflammation. Lupeol can potentially serve as a novel therapeutic agent against visceral leishmaniasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Leishmania infantum belongs to the L. donovani complex, which includes species associated with visceral leishmaniasis. Traditionally, antimonial compounds have served as the primary antiparasitic treatment for all clinical forms of leishmaniasis. However, the global spread of resistance to these compounds has posed a significant challenge in the treatment in some regions. In this study, we aimed to investigate resistance to trivalent sodium stibogluconate in vitro using promastigotes from a wild strain of L. infantum. We compared the growth rates and proteomic profiles of wild-type and resistant line conducting label-free quantitative mass spectrometry-based proteomic analyses. Statistical and bioinformatics analyses were employed to evaluate the significance of protein concentration changes, protein identity annotation, GO term analysis, biosynthetic pathways, and protein-protein interactions. Our findings revealed that the resistant line displayed a notable reduction in growth rate. Proteomic data unveiled similar protein concentrations per cell in both groups but with differing molecule copy numbers. We identified 165 proteins with increased concentration, these were associated with transcription and translation activities, lipid metabolism, energy metabolism, and peroxisome biogenesis. In the decreased protein groups were 56 proteins linked to metal acquisition and metabolism, particularly iron. These results suggest a novel perspective on antimonial resistance, highlighting the importance of post-transcriptional and post-translational regulation, alongside energy expenditure compensation and alterations in organelle membrane lipid composition in antimonial-resistant parasites. Overall, our study provides insights into the proteomic profile of stibogluconate-resistant strain, contributing to our general understanding of the complex landscape of antiparasitic resistance in L. infantum. SIGNIFICANCE: Species within the Leishmania donovani complex are implicated in cases of visceral leishmaniasis in the world. Leishmania infantum is a species that predominates in regions spanning the Mediterranean Basin, the Middle East, Central Asia, South and Central America. Antimonials were the first treatment for leishmaniasis, however in the last decades, the resistance has emerged in subregions like India, where it is not a therapeutic option. In contrast, sodium stibogluconate (SbIII) remains the first-line treatment in the Americas. Unfortunately, the emergence of resistance has outpaced the development of new therapeutic options, thereby becoming a critical point in the struggle against the disease. In this study we performed an in-depth proteomic analysis with liquid chromatography mass-mass spectrometry (LC-MS/MS) on L. infantum with Sb-induced resistance in vitro. Results showed a complex proteomic adaptation in the resistant line, involving transcriptional and translational proteins, energy compensation, and homeostasis maintenance. These insights contribute to understanding the molecular adaptation in the parasite and provide information to new investigations related to therapeutics development., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Macrophages are major host cells for the protozoan Leishmania parasite. Depending on their activation state, they either contribute to the detection and elimination of Leishmania spp. or promote parasite resilience. Here, we report that the activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in macrophages plays a pivotal role in the progression of Leishmania infantum infection by controlling inflammation and redox balance of macrophages. We also highlight the involvement of the NOX2/reactive oxygen species (ROS) axis in early Nrf2 activation and, subsequently, prostaglandin E2 (PGE2)/EP2r signaling in the sustenance of Nrf2 activation upon infection. Moreover, we establish a ferroptosis-like process within macrophages as a cell death program of L. infantum and the protective effect of Nrf2 in macrophages against L. infantum death. Altogether, these results identify Nrf2 as a critical factor for the susceptibility of L. infantum infection, highlighting Nrf2 as a promising pharmacological target for the development of therapeutic approaches for the treatment of visceral leishmaniasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Obligate intracellular protozoan parasite, Leishmania donovani, causative agent of visceral leishmaniasis, led to impaired macrophage functions. It is well documented that many of these changes were induced by parasite-mediated reduction in macrophage cholesterol content. Leishmania-mediated alteration in the other lipids has not been explored in detail yet. Here, we found that the expression of key cholesterol biosynthetic genes and total cellular cholesterol were reduced during L. donovani infection. Further, we have also identified that this reduction in the cholesterol led to increased membrane fluidity and inhibition of antigen-presenting potential of macrophages. In addition to this, we studied the relative changes in different lipids in THP-1-derived macrophages during L. donovani infection through liquid chromatography-mass spectrometry. We found that Sphingomyelin (16:0) and ceramide (20:1, 26:0 and 26:1) were significantly reduced in infected macrophages. We further observed that the majority of different sub-classes of phospholipids were downregulated significantly. Overall ratio of phosphatidylcholine versus phosphotidylethanolamine was decreased which indicated the compensatory mechanism of cell in response to cholesterol reduction. The observed Leishmania-mediated alteration in macrophage-lipidome provided the novel insights into mechanism of host-pathogen interactions., (© 2024 John Wiley & Sons Ltd.)

Cullin-1-RING ubiquitin ligases (CRL1) or SCF1 (SKP1-CUL1-RBX1) E3 ubiquitin ligases are the largest and most extensively investigated class of E3 ligases in mammals that regulate fundamental processes, such as the cell cycle and proliferation. These enzymes are multiprotein complexes comprising SKP1, CUL1, RBX1, and an F-box protein that acts as a specificity factor by interacting with SKP1 through its F-box domain and recruiting substrates via other domains. E3 ligases are important players in the ubiquitination process, recognizing and transferring ubiquitin to substrates destined for degradation by proteasomes or processing by deubiquitinating enzymes. The ubiquitin-proteasome system (UPS) is the main regulator of intracellular proteolysis in eukaryotes and is required for parasites to alternate hosts in their life cycles, resulting in successful parasitism. Leishmania UPS is poorly investigated, and CRL1 in L. infantum, the causative agent of visceral leishmaniasis in Latin America, is yet to be described. Here, we show that the L. infantum genes LINF_110018100 (SKP1-like protein), LINF_240029100 (cullin-like protein-like protein), and LINF_210005300 (ring-box protein 1 -putative) form a LinfCRL1 complex structurally similar to the H. sapiens CRL1. Mass spectrometry analysis of the LinfSkp1 and LinfCul1 interactomes revealed proteins involved in several intracellular processes, including six F-box proteins known as F-box-like proteins (Flp) (data are available via ProteomeXchange with identifier PXD051961). The interaction of LinfFlp 1-6 with LinfSkp1 was confirmed, and using in vitro ubiquitination assays, we demonstrated the function of the LinfCRL1(Flp1) complex to transfer ubiquitin. We also found that LinfSKP1 and LinfRBX1 knockouts resulted in nonviable L. infantum lineages, whereas LinfCUL1 was involved in parasite growth and rosette formation. Finally, our results suggest that LinfCul1 regulates the S phase progression and possibly the transition between the late S to G2 phase in L. infantum. Thus, a new class of E3 ubiquitin ligases has been described in L. infantum with functions related to various parasitic processes that may serve as prospective targets for leishmaniasis treatment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rolemberg Santana Travaglini Berti de Correia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Host anti-inflammatory responses are critical for the progression of visceral leishmaniasis, and the pleiotropic cytokine interleukin (IL)-33 was found to be upregulated in infection. Here, we documented that IL-33 induction is a consequence of elevated cAMP-mediated exchange protein activated by cAMP (EPAC)/calcineurin-dependent signaling and essential for the sustenance of infection. Leishmania donovani-infected macrophages showed upregulation of IL-33 and its neutralization resulted in decreased parasite survival and increased inflammatory responses. Infection-induced cAMP was involved in IL-33 production and of its downstream effectors PKA and EPAC, only the latter was responsible for elevated IL-33 level. EPAC initiated Rap-dependent phospholipase C activation, which triggered the release of intracellular calcium followed by calcium/calmodulin complex formation. Screening of calmodulin-dependent enzymes affirmed involvement of the phosphatase calcineurin in cAMP/EPAC/calcium/calmodulin signaling-induced IL-33 production and parasite survival. Activated calcineurin ensured nuclear localization of the transcription factors, nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha required for IL-33 transcription, and we further confirmed this by chromatin immunoprecipitation assay. Administering specific inhibitors of nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha in BALB/c mouse model of visceral leishmaniasis decreased liver and spleen parasite burden along with reduction in IL-33 level. Splenocyte supernatants of inhibitor-treated infected mice further documented an increase in tumor necrosis factor alpha and IL-12 level with simultaneous decrease of IL-10, thereby indicating an overall disease-escalating effect of IL-33. Thus, this study demonstrates that cAMP/EPAC/calcineurin signaling is crucial for the activation of IL-33 and in effect creates anti-inflammatory responses, essential for infection., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Berardinelli-Seip congenital lipodystrophy (CGL), a rare autosomal recessive disorder, is characterized by a lack of adipose tissue. Infections are one of the major causes of CGL individuals' premature death. The mechanisms that predispose to infections are poorly understood. We used Leishmania infantum as an in vitro model of intracellular infection to explore mechanisms underlying the CGL infection processes, and to understand the impact of host mutations on Leishmania survival, since this pathogen enters macrophages through specialized membrane lipid domains. The transcriptomic profiles of both uninfected and infected monocyte-derived macrophages (MDMs) from CGL (types 1 and 2) and controls were studied. MDMs infected with L. infantum showed significantly downregulated expression of genes associated with infection-response pathways (MHC-I, TCR-CD3, and granzymes). There was a transcriptomic signature in CGL cells associated with impaired membrane trafficking and signaling in response to infection, with concomitant changes in the expression of membrane-associated genes in parasites (e.g. δ-amastins). We identified pathways suggesting the lipid storage dysfunction led to changes in phospholipids expression and impaired responses to infection, including immune synapse (antigen presentation, IFN-γ signaling, JAK/STAT); endocytosis; NF-kappaB signaling; and phosphatidylinositol biosynthesis. In summary, lipid metabolism of the host plays an important role in determining antigen presentation pathways., (© 2024. The Author(s).)

Towards identification of novel therapeutic candidates, a series of quinazolinone-based acetamide derivatives were synthesized and assessed for their anti-leishmanial efficacy. Amongst synthesized derivatives, compounds F12, F27 and F30 demonstrated remarkable activity towards intracellular L. donovani amastigotes in vitro, with IC 50 values of 5.76 ± 0.84 μM, 3.39 ± 0.85 μM and 8.26 ± 1.23 μM against promastigotes, and 6.02 μM ± 0.52, 3.55 ± 0.22 μM and 6.23 ± 0.13 μM against amastigotes, respectively. Oral administration of compounds F12 and F27 entailed >85% reduction in organ parasite burden in L. donovani-infected BALB/c mice and hamsters, by promoting host-protective Th1 cytokine response. In host J774 macrophages, mechanistic studies revealed inhibition of PI3K/Akt/CREB axis, resulting in a decrease of IL-10 versus IL-12 release upon F27 treatment. In silico docking studies conducted with lead compound, F27 demonstrated plausible inhibition of Leishmania prolyl-tRNA synthetase, which was validated via detection of decreased proline levels in parasites and induction of amino acid starvation, leading to G 1 cell cycle arrest and autophagy-mediated programmed cell death of L. donovani promastigotes. Structure-activity analysis and study of pharmacokinetic and physicochemical parameters suggest oral availability and underscore F27 as a promising lead for anti-leishmanial drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excreted in canine sera from dogs with canine visceral leishmaniasis (CanVL). A total of 56 canine sera were divided into Group I (28, from healthy dogs) and Group II (28, from the same dogs, but already with CanVL). CanVL was determined by clinical and laboratory diagnoses. Canine sera were ultra-centrifuged to recover EVs (Can-EVs). Analyses by transmission electron microscopy, nanoparticle tracking analysis (NTA), sodium dodecyl sulfate-poli-acrylammide gel eletroforesis (SDS-PAGE) and, Immunoblot confirmed the presence of (i) microvesicles/exosomes and (ii) the tetraspanins CD63 and CD9. EVs secreted by Leishmania (Leishmania) infantum-EVs were reactive against sera from dogs with CanVL (performed by ELISA and Immunoblot). NTA analyses exhibited that concentrations of Can-EVs from dogs with CanVL (7.78 × 10 10 Can-EVs/mL) were higher (p < .0001) than the non-infected dogs (mean: 1.47 × 10 10 Can-EVs/mL). These results suggested that concentrations of Can-EVs were able to distinguish dogs with CanVL from healthy dogs. The relative expressions of 11 miRNAs species (miR-21-5p, miR-146a-5p, miR-125b-5p, miR-144-3p, miR-194-5p, miR-346, miR-29c-3p, miR-155-5p, miR-24-3p, miR-181a-5p, and miR-9-5p) were estimated in purified miRNAs of 30 canine sera. Dogs with CanVL up-expressed miR-21-5p and miR-146a-5p when compared with healthy dogs. The other miRNA species were poorly or not expressed in canine sera. In conclusion, this study suggests that CanVL induces changes in size and concentration of Can-EVs, as well as, the up-expression of miR-21-5p and miR-146a-5p in infected dogs., (© 2023 John Wiley & Sons Ltd.)

Leishmania donovani infection of macrophages drives profound changes in the metabolism of both the host macrophage and the parasite, which undergoes different phases of development culminating in replication and propagation. However, the dynamics of this parasite-macrophage cometabolome are poorly understood. In this study, a multiplatform metabolomics pipeline combining untargeted, high-resolution CE-TOF/MS and LC-QTOF/MS with targeted LC-QqQ/MS was followed to characterize the metabolome alterations induced in L. donovani -infected human monocyte-derived macrophages from different donors at 12, 36, and 72 h post-infection. The set of alterations known to occur during Leishmania infection of macrophages, substantially expanded in this investigation, characterized the dynamics of the glycerophospholipid, sphingolipid, purine, pentose phosphate, glycolytic, TCA, and amino acid metabolism. Our results showed that only citrulline, arginine, and glutamine exhibited constant trends across all studied infection time points, while most metabolite alterations underwent a partial recovery during amastigote maturation. We determined a major metabolite response pointing to an early induction of sphingomyelinase and phospholipase activities and correlated with amino acid depletion. These data represent a comprehensive overview of the metabolome alterations occurring during promastigote-to-amastigote differentiation and maturation of L. donovani inside macrophages that contributes to our understanding of the relationship between L. donovani pathogenesis and metabolic dysregulation.

To initiate an antileishmanial adaptive immune response, dendritic cells (DCs) must carry Leishmania antigens from peripheral tissues to local draining lymph nodes. However, the migratory capacity of DCs is largely compromised during Leishmania donovani infection. The molecular mechanism underlying this defective DC migration is not yet fully understood. Here, we demonstrate that L. donovani infection impaired the lymph node homing ability of DCs by decreasing C-type lectin receptor 2 (CLEC-2) expression. L. donovani exerted this inhibitory effect by inducing transforming growth factor-β (TGF-β) secretion from DCs. Indeed, TGF-β produced in this manner inhibited nuclear factor-κB (NF-κB)-mediated CLEC-2 expression on DCs by activating c-Src. Notably, suppression of c-Src expression significantly improved the arrival of DCs in draining lymph nodes by preventing L. donovani-induced CLEC-2 downregulation on DCs. These findings reveal a unique mechanism by which L. donovani inhibits DC migration to lymph nodes and suggest a key role for TGF-β, c-Src, and CLEC-2 in regulating this process. IMPORTANCE Dendritic cells (DCs) play a key role in initiating T cell-mediated protective immunity against visceral leishmaniasis (VL), the second most lethal parasitic disease in the world. However, the T cell-inducing ability of DCs critically depends on the extent of DC migration to regional lymph nodes. Notably, the migration of DCs is reported to be impaired during VL. The cause of this impaired DC migration, however, remains ill-defined. Here, we provide the first evidence that L. donovani, the causative agent of VL, attenuates the lymph node homing capacity of DCs by decreasing C-type lectin receptor 2 (CLEC-2) expression on DCs. Additionally, we have demonstrated how L. donovani mediates this inhibitory effect. Overall, our work has revealed a unique mechanism underlying L. donovani-induced impairment of DC migration and suggests a potential strategy to improve antileishmanial T cell activity by increasing DC arrival in lymph nodes., Competing Interests: The authors declare no conflict of interest.

Visceral leishmaniasis (VL) is a progressive, debilitating, and potentially fatal disease if left untreated. As a neglected tropical disease (NTD), the available treatment is restricted to a few drugs, which typically must be administered over a long period but are associated with serious adverse effects and have variability in efficacy. In this sense, drug repositioning has been considered an excellent strategy in the search for alternative treatments, especially in reducing the time and cost of the research. In this work, the repositioning potential of amodiaquine (AQ), a well-known antimalarial drug, was investigated for the treatment of VL. AQ showed significant and selective activity against promastigotes (IC 50  = 11.6 μg/mL) and intracellular amastigotes (IC 50  = 2.4 μg/mL) of L. infantum, being 10 times more destructive to the intracellular parasites than the host cell. In addition, pre-treatment of macrophages with AQ caused a significant reduction in the infection index, indicating a prophylactic effect of this drug. SEM images showed that AQ induces strong shape alterations of the promastigotes with an increase in cell volume with rounding and ribbing (vertical ridges), as well as a shortened flagellum. In addition, AQ induced depolarization of the ΔΨm, an increase in ROS and neutral lipids levels, and changes in the cell cycle in promastigotes, without alterations to the permeability of the parasite plasma membrane. L. infantum-infected macrophages treated with AQ induced the activation of oxidative mechanisms by infected host cells, with an increase in ROS and NO levels. Finally, in vitro interactions between AQ and miltefosine were found to have an additive effect in both biological stages of the parasite, with the ∑FIC 50 values ranging from 0.74 to 1.16 μg/mL and 0.54-1.11 μg/mL for promastigotes and intracellular amastigotes, respectively. Overall, these data highlight the utility of drug repurposing and indicate future preclinical testing for AQ itself or in combination as a potential VL treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Vector-borne infections of humans with the protozoan parasite Leishmania (L.) infantum can cause a systemic and potentially lethal disease termed visceral leishmaniasis. In the corresponding mouse model, an intravenous infection with L. infantum leads to the persistence of parasites in various organs, including bone marrow (BM). Considering the anatomical proximity between the BM and the cortical bone, we investigated whether a chronic infection with L. infantum affected bone homeostasis. Unexpectedly, chronic infection with L. infantum caused an increase in bone mass in mice. In vivo, an increased number of osteoblasts and osteocytes and a decreased maturation of osteoclasts characterized the phenotype. Confocal laser scanning fluorescence microscopy confirmed the infection of BM macrophages but also revealed the presence of parasites in osteoclasts. In vitro, mature osteoclasts took up L. infantum parasites. However, infection of osteoclast progenitors abolished their differentiation and function. In addition, secretory products of infected BM-derived macrophages inhibited the maturation of osteoclasts. Both in vitro and in vivo, infected macrophages and osteoclasts showed an enhanced expression of the anti-osteoclastogenic chemokine CCL5 (RANTES). Neutralization of CCL5 prevented the inhibition of osteoclast generation seen in the presence of culture supernatants from L. infantum-infected macrophages. Altogether, our study shows that chronic infection with Leishmania increases bone mass by inducing bone formation and impairing osteoclast differentiation and function. © 2022 American Society for Bone and Mineral Research (ASBMR)., (© 2022 American Society for Bone and Mineral Research (ASBMR).)

Infection with Leishmania donovani reduces cellular cholesterol and thus deprives the host cells by inhibiting its synthesis and uptake. Changes in cholesterol levels increase the chance of attachment and internalization of L. donovani in macrophages (Mϕ). Retinoic acid (RA), an important micronutrient, restores the lysosomal uptake of cholesterol in L. donovani-infected Mϕ. Importantly, mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) increases the cellular cholesterol level by increasing expression of sterol regulatory element-binding protein 2 ( SREBP2 ). Whether the efficacy of RA in L. donovani-infected Mϕ is mediated by mTOR is not yet established. Moreover, there are contradicting reports suggesting potential activation and inhibition of mTOR in L. donovani-infected Mϕ. Intrigued by this, we attempted to understand the RA-mediated restoration of cholesterol as well as the possible roles of mTORC1, if any. Our findings suggest that L. donovani infection impairs the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), uptake of low-density lipoprotein receptor (LDLR), and secretion of ATP-binding cassette transporter (ABCA1) in Mϕ. L. donovani infection possibly impairs mTORC1 formation, as it inhibits the expression of regulatory-associated protein of mammalian target of rapamycin ( RAPTOR ). Importantly, all these are restored upon RA supplementation. RA also restores the levels of SREBP2 in L. donovani-infected Mϕ, resulting in increased cellular cholesterol and thus reducing the parasite burden. When mTORC1 was inhibited, RA exerted a similar response in L. donovani-infected Mϕ; i.e., it restored cholesterol levels and reduced the parasite burden. In summary, RA restores cholesterol levels in L. donovani-infected Mϕ and reduces the parasite burden in an mTOR-independent manner. IMPORTANCE People who reside in regions where leishmaniasis is endemic and who lack proteins, iron, zinc, and vitamin A in their diet are more prone to develop visceral leishmaniasis (VL) as a full-blown disease. Vitamin A deficiency favors the development of a parasitic infection in the human host, and the WHO recommends administering 200,000-IU doses to VL patients on admission. Additionally, Leishmania entry and its survival inside the host are achieved by utilizing host cholesterol, as all trypanosomatids lack de novo synthesis of sterol. We have already shown that RA regulates cellular cholesterol levels associated with an efficient immune response. A deficiency of retinoic acid (RA) favors the parasite in Leishmania donovani-infected macrophages by downregulating the immune response. In the present work, we observed that RA restores cellular cholesterol levels in Leishmania donovani-infected macrophages. This study proposes using RA as an immune potentiator along with standard therapy.

Leishmaniasis is considered a tropical neglected disease, which is caused by an intramacrophagic parasite, Leishmania. It is endemic in 89 different countries. Autophagy-related protein 8 (Ldatg8) is responsible for the transformation of parasites from promastigote to amastigote differentiation. Ldatg8 is one of the key drug targets of Leishmania donovani (L. donovani) responsible for the defense of parasites during stress conditions. Virtual screening of natural ligand library had been performed against Ldatg8 to identify novel and potent inhibitors. Molecular docking and molecular dynamics simulation studies showed that urolithin A stably blocked Ldatg8. Urolithins are combinations of coumarin and isocoumarin. Further, we evaluated the antileishmanial effects of urolithin A by antileishmanial assays. Urolithin A inhibited the growth and proliferation of L. donovani promastigotes with an IC 50  value of 90.3 ± 6.014 μM. It also inhibited the intramacrophagic parasite significantly with an IC 50  value of 78.67 ± 4.62 μM. It showed limited cytotoxicity to the human THP-1 differentiated macrophages with a CC 50  value of 190.80 ± 16.89 μM. Further, we assayed reactive oxygen species (ROS) generation and annexin V/PI staining upon urolithin A treatment of parasites to have an insight into the mechanism of its action. It induced ROS significantly in a dose-dependent manner, which caused apoptosis partially in parasites. The potential inhibitors for Ldatg8, identified in this study, would provide the platform for the development of an effective and affordable antileishmanial drug., (© 2022 John Wiley & Sons Ltd.)

Visceral leishmaniasis (VL) is often associated with hematologic manifestations that may interfere with neutrophil response. Lipophosphoglycan (LPG) is a major molecule on the surface of Leishmania promastigotes, which has been associated with several aspects of the parasite-vector-host interplay. Here, we investigated how LPG from Leishmania ( L .) infantum , the principal etiological agent of VL in the New World, influences the initial establishment of infection during interaction with human neutrophils in an experimental setting in vitro . Human neutrophils obtained from peripheral blood samples were infected with either the wild-type L. infantum (WT) strain or LPG-deficient mutant ( ∆lpg1 ). In this setting, ∆lpg1 parasites displayed reduced viability compared to WT L. infantum ; such finding was reverted in the complemented ∆lpg1 + LPG1 parasites at 3- and 6-h post-infection. Confocal microscopy experiments indicated that this decreased survival was related to enhanced lysosomal fusion. In fact, LPG-deficient L. infantum parasites more frequently died inside neutrophil acidic compartments, a phenomenon that was reverted when host cells were treated with Wortmannin. We also observed an increase in the secretion of the neutrophil collagenase matrix metalloproteinase-8 (MMP-8) by cells infected with ∆lpg1 L. infantum compared to those that were infected with WT parasites. Furthermore, collagen I matrix degradation was found to be significantly increased in ∆lpg1 parasite-infected cells but not in WT-infected controls. Flow cytometry analysis revealed a substantial boost in production of reactive oxygen species (ROS) during infection with either WT or ∆lpg1 L. infantum . In addition, killing of ∆lpg1 parasites was shown to be more dependent on the ROS production than that of WT L. infantum . Notably, inhibition of the oxidative stress with Apocynin potentially fueled ∆lpg1 L. infantum fitness as it increased the intracellular parasite viability. Thus, our observations demonstrate that LPG may be a critical molecule fostering parasite survival in human neutrophils through a mechanism that involves cellular activation and generation of free radicals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Quintela-Carvalho, Goicochea, Mançur-Santos, Viana, Luz, Dias, Lázaro-Souza, Suarez, de Oliveira, Saraiva, Brodskyn, Veras, de Menezes, Andrade, Lima, Descoteaux and Borges.)

Recently unfolded mechanisms showed lipid droplet helps in pathogen survival and paralyzes host immune response. In the present study, we showed the extent of lipid droplet(LD) generation in Leishmania donovani infection, the signaling involved, and their function concerning pathogenicity. RAW 264.7 and J774A.1 cells were used to infect with L. donovani and then flow cytometry and confocal microscopy were used to detect lipid droplet generation and subsequent assays. In this study, we showed that L. donovani AG83 (AG83/MHOM/1983) triggers lipid droplet formation in macrophages in a time-dependent manner. We provide novel insight into the signaling molecules which is responsible for LD accumulation. Interestingly, LPG deficient attenuated Leishmania strain UR6 (UR6/MHOM/1978) failed to fuel LD generation. But inhibition of phagosome maturation drastically stimulates LD accumulation in UR6 infected MΦs. Aspirin treatment in AG83 infected MΦs does not only lower LD load but also favors phagolysosome biogenesis and corrects cytokine balance. Employing strategies to circumvent halt in phagosome maturation using drugs that manipulate lipid droplet generation could be used as a therapeutic tool to resist parasite growth in the early hour of infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This study compared the therapeutic potential of the chemotherapy using meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes (Nano Sb v ) and immunotherapy with anti-canine IL-10 receptor-blocking monoclonal antibody (Anti IL-10R) on canine visceral leishmaniasis (CVL). Twenty mongrel dogs naturally infected by L. infantum, displaying clinical signs of visceral leishmaniasis were randomly divided in two groups. In the first one, nine dogs received six intravenous doses of a mixture of conventional and PEGylated liposomes containing meglumine antimoniate at 6.5 mg Sb/kg/dose. In the second one, eleven dogs received two intramuscular doses of 4 mg of anti-canine IL-10 receptor-blocking monoclonal antibody. The animals were evaluated before (T0) and 30, 90, and 180 days after treatments. Our major results demonstrated that both treatments were able to maintain hematological and biochemical parameters, increase circulating T lymphocytes subpopulations, increase the IFN-γ producing T-CD4 lymphocytes, restore the lymphoproliferative capacity and improve the clinical status. However, although these improvements were observed in the initial post-treatment times, they did not maintain until the end of the experimental follow-up. We believe that the use of booster doses or the association of chemotherapy and immunotherapy (immunochemotherapy) is promising to improve the effectiveness of treating CVL for improving the clinical signs and possibly reducing the parasite burden in dogs infected with Leishmania infantum., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Increasing evidence suggests that in hosts infected with parasites of the Leishmania donovani complex, transmission of infection to the sand fly vector is linked to parasite repositories in the host skin. However, a detailed understanding of the dispersal (the mechanism of spread) and dispersion (the observed state of spread) of these obligatory-intracellular parasites and their host phagocytes in the skin is lacking. Using endogenously fluorescent parasites as a proxy, we apply image analysis combined with spatial point pattern models borrowed from ecology to characterize dispersion of parasitized myeloid cells (including Man R+ and CD11c + cells) and predict dispersal mechanisms in a previously described immunodeficient model of L. donovani infection. Our results suggest that after initial seeding of infection in the skin, heavily parasite-infected myeloid cells are found in patches that resemble innate granulomas. Spread of parasites from these initial patches subsequently occurs through infection of recruited myeloid cells, ultimately leading to self-propagating networks of patch clusters. This combination of imaging and ecological pattern analysis to identify mechanisms driving the skin parasite landscape offers new perspectives on myeloid cell behavior following parasitism by L. donovani and may also be applicable to elucidating the behavior of other intracellular tissue-resident pathogens and their host cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Doehl, Ashwin, Brown, Romano, Carmichael, Pitchford and Kaye.)

Interactions of Leishmania donovani secretory virulence factors with the host proteins and their interplay during the infection process in humans is poorly studied in Visceral Leishmaniasis. Lack of a holistic study of pathway level de-regulations caused due to these virulence factors leads to a poor understanding of the parasite strategies to subvert the host immune responses, secure its survival inside the host and further the spread of infection to the visceral organs. In this study, we propose a computational workflow to predict host-pathogen protein interactome of L.donovani secretory virulence factors with human proteins combining sequence-based Interolog mapping and structure-based Domain Interaction mapping techniques. We further employ graph theoretical approaches and shortest path methods to analyze the interactome. Our study deciphers the infection paths involving some unique and understudied disease-associated signaling pathways influencing the cellular phenotypic responses in the host. Our statistical analysis based in silico knockout study unveils for the first time UBC, 1433Z and HS90A mediator proteins as potential immunomodulatory candidates through which the virulence factors employ the infection paths. These identified pathways and novel mediator proteins can be effectively used as possible targets to control and modulate the infection process further aiding in the treatment of Visceral Leishmaniasis., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Visceral leishmaniasis (VL) or Kala-azar is a vector borne protozoan infection caused by Leishmania donovani in the Indian subcontinent mainly India, Nepal and Bangladesh. It is a major public health problem in these countries mostly affecting the socio-economically poor population. Leishmaniasis ranks the third most important disease after malaria and filariasis but is still considered as one of the neglected tropical diseases of the world. For development of better therapeutic agents and effective vaccine against VL, there is a need to understand host immunological changes that play a vital role during course of infection. Therefore, we investigated the role of Th17 pathway in Balb/c mice during Leishmania donovani infection and treatment with amphotericin B. Mice were divided in four groups i.e. Control, Infected, Uninfected treated and Infected treated. The cytokine levels were estimated in the spleen of Balb/c mice on days 1, 3, 7, 14, 17, 21, 28, 35, 45 and 60 post infection and during course of treatment. The mRNA levels of the Th17 pathway during active Leishmania donovani infection and after treatment were determined by real time polymerase chain reaction (RT-PCR) and protein levels by flow cytometry and ELISA. Results of our study revealed that active infection was associated with low levels of Th17 cytokines IL-17, IL-22 and IL-23 and elevated levels of IL-6, IL-1β and TGF-β. Amphotericin B treatment restored production of pro-inflammatory cytokines IL-17 and IL-22. The levels of transcription factor RORγt were found to correlate with the levels of IL-17 during infection and also after chemotherapy whereas STAT3 levels were elevated during infection and vice versa after treatment. The findings of this study suggest that Th17 cytokines IL-17 and IL-22 are associated with protection against VL infection and development of any interventions or chemotherapeutic agents targeting Th17 pathway could be an important approach for VL treatment., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Visceral leishmaniasis (VL) is a chronic and often fatal disease caused by protozoans of the genus Leishmania that affects millions of people worldwide. Patients with symptomatic VL have an impaired anti- Leishmania -specific CD4 + T-cell response, which is reversed after clinical cure. In contrast, the quality of the CD4 + and CD8 + T-cell responses involved in resistance and/or cure of VL relies on the capability of these cells to activate polyfunctional and memory responses, which are associated with the simultaneous production of three cytokines: IFN-γ, IL-2, and TNF-α. Models for the development of CD4 and CD8 T-cell quality in memory and protection to leishmaniasis have been described previously. We aimed to assess the functionality of the T cells involved in the recovery of the immune suppression throughout the VL treatment. Therefore, we cultured peripheral blood mononuclear cells (PBMCs) from VL patients and healthy controls in vitro with soluble Leishmania antigen (SLA). Cell surface markers and intracellular cytokine production were determined on days 7, 14, 21, 30, 60, 90, and 180 after the beginning of chemotherapy. We observed that the frequencies of CD4 + TNF-α + IFN-γ + and the multifunctional CD4 + IL-2 + TNF-α + IFN-γ + , together with CD4 + TNF-α + and CD4 + IFN-γ + T cells, increased throughout and at the end of the treatment, respectively. In addition, enhanced frequencies of CD8 + IL-2 + TNF-α + IFN-γ + and CD8 + TNF-α + IFN-γ T cells were also relevant in the healing process. Noteworthy, the frequencies of the CD4 + and CD8 central-memory T cells, which produce IL-2, TNF-α, and IFN-γ and ensure the memory response against parasite reinfection, are significantly enhanced in cured patients. In addition, the subset of the non-functional CD8 Low population is predominant in VL untreated patients and decreases along the chemotherapy treatment. In contrast, a CD8 High subset increased towards the cure. Furthermore, the cure due to treatment with meglumine antimoniate or with liposomal amphotericin B was associated with the recovery of the T-cell immune responses. We described the evolution and participation of functional T cells during the treatment of patients with VL. Our results disclosed that the clinical improvement of patients is significantly associated with the participation of the CD4 + and CD8 + cytokine-secreting T cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rodrigues, Barreto, Bomfim, Gomes, Ferreira, da Cruz, Magalhães, de Jesus, Palatnik-de-Sousa, Corrêa and de Almeida.)

Leishmaniasis is a neglected parasitic disease for which the conventional treatment can be considered inefficient and extremely aggressive, generating several and severe side effects. Therefore, the discovery of new drug candidates is important for the improvement in the quality of life of patients. Previously, we reported the promising results of isopentyl caffeate (ICaf) against Leishmania chagasi (agent of visceral leishmaniasis) and Leishmania amazonensis (agent of cutaneous leishmaniasis) promastigotes, displaying IC 50 of 1.56 and 1.71 μM, respectively. Herein, we aimed to decipher the mechanisms of anti-Leishmania action of ICaf. Light and scanning electron microscopy assays showed relevant morphological changes in promastigotes when treated with ICaf, including rounding of the parasite body, shortening of the flagellum, blebs on the plasma membrane and cellular aggregation. The parasite mitochondrion was targeted by ICaf, resulting in a significant reduction in its metabolic activity and electric membrane potential followed by an increase in the production of reactive oxygen species, which culminated in the loss of plasma membrane integrity and parasite death. Relevantly, ICaf also had a potent anti-amastigote action. The IC 50 values calculated for intracellular amastigotes of L. amazonensis were 3.27, 1.60 and 1.52 μM, while for L. chagasi the values were 2.48, 1.84 and 1.60 μM, respectively, after treating the infected macrophages with ICaf for 24, 48 and 72 h. ICaf was well tolerated by THP-1 macrophages, which gave rise to excellent selectivity indexes considering both Leishmania species. The current results suggest that ICaf may emerge as a chemotherapeutic alternative for the treatment of leishmaniasis., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Intracellular phagosomal pathogens represent a formidable challenge for innate immune cells, as, paradoxically, these phagocytic cells can act as both host cells that support pathogen replication and, when properly activated, are the critical cells that mediate pathogen elimination. Infection by parasites of the Leishmania genus provides an excellent model organism to investigate this complex host-pathogen interaction. In this review we focus on the dynamics of Leishmania amazonensis infection and the host innate immune response, including the impact of the adaptive immune response on phagocytic host cell recruitment and activation. L. amazonensis infection represents an important public health problem in South America where, distinct from other Leishmania parasites, it has been associated with all three clinical forms of leishmaniasis in humans: cutaneous, muco-cutaneous and visceral. Experimental observations demonstrate that most experimental mouse strains are susceptible to L. amazonensis infection, including the C57BL/6 mouse, which is resistant to other species such as Leishmania major , Leishmania braziliensis and Leishmania infantum . In general, the CD4 + T helper (Th)1/Th2 paradigm does not sufficiently explain the progressive chronic disease established by L. amazonensis , as strong cell-mediated Th1 immunity, or a lack of Th2 immunity, does not provide protection as would be predicted. Recent findings in which the balance between Th1/Th2 immunity was found to influence permissive host cell availability via recruitment of inflammatory monocytes has also added to the complexity of the Th1/Th2 paradigm. In this review we discuss the roles played by innate cells starting from parasite recognition through to priming of the adaptive immune response. We highlight the relative importance of neutrophils, monocytes, dendritic cells and resident macrophages for the establishment and progressive nature of disease following L. amazonensis infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Carneiro and Peters.)

Considering the collaboration between immune responses and medications for the improvement of visceral leishmaniasis (VL), this study investigated the levels of T helper (Th) 22 and the corresponding cytokines in the acute phase of VL and their alterations following treatment. The study was conducted on 18 patients with confirmed VL and 20 healthy sex and age matched children as the controls. The levels of Th22 cells and the cytokines driving their differentiations and functions in the blood and peripheral blood mononuclear cells (PBMC) cultured supernatants, were assessed using flow cytometry method. The results revealed significantly higher levels of Th22, IL-21 and IL-6 in the patients' blood than those in the controls. Additionally, higher levels of IL-21 and IL-22 were observed in the cultured supernatants of VL patients' PBMCs, compared to the controls. Upon treatment, Th22 and IL-6 were down-regulated and conversely, IL-21, IL-22, IL-23 and IL-33 were significantly up-regulated in the patients' blood at different time points. Receiver operating characteristic (ROC) curve analysis indicated that for the differential diagnosis of VL, plasma IL-21 is more sensitive and specific than Th22 and the above mentioned cytokines. The higher proportions of Th22 and IL-21 in the VL patients and their alterations post treatment confer their roles in the immunopathogenesis of VL. So, Th22 and IL-21 in the patients' blood can be considered as biomarkers to be used for the differential diagnosis of VL. Nevertheless, further studies are warranted to clarify their particular mechanisms in this process., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Transcriptional analysis of complex biological scenarios has been used extensively, even though sometimes the results of such analysis may prove imprecise or difficult to interpret due to an overwhelming amount of information. In this study, a large-scale real-time qPCR experiment was coupled to multivariate statistical analysis in order to describe the main immunological events underlying the early L. infantum infection in livers of BALB/c mice. High-throughput qPCR was used to evaluate the expression of 223 genes related to immunological response and metabolism 1, 3, 5, and 10 days post infection. This integrative analysis showed strikingly different gene signatures at 1 and 10 days post infection, revealing the progression of infection in the experimental model based on the upregulation of particular immunological response patterns and mediators. The gene signature 1 day post infection was not only characterized by the upregulation of mediators involved in interferon signaling and cell chemotaxis, but also the upregulation of some inhibitory markers. In contrast, at 10 days post infection, the upregulation of many inflammatory and Th1 markers characterized a more defined gene signature with the upregulation of mediators in the IL-12 signaling pathway. Our results reveal a significant connection between the expression of innate immune response and metabolic and inhibitory markers in early L. infantum infection of the liver.

We showed previously that antioxidant enzyme heme oxygenase 1 (HO-1) is critical for Leishmania survival in visceral leishmaniasis. HO-1 inhibits host oxidative burst and inflammatory cytokine production, leading to parasite persistence. In the present study, screening of reported HO-1 transcription factors revealed that infection upregulated (4.1-fold compared to control [ P  < 0.001]) nuclear factor erythroid 2 (NFE2)-related factor 2 (NRF2). Silencing of NRF2 reduced both HO-1 expression and parasite survival. Investigation revealed that infection-induced transient reactive oxygen species (ROS) production dissociated NRF2 from its inhibitor KEAP1 and enabled phosphorylation-dependent nuclear translocation. Both NRF2 and HO-1 silencing in infection increased production of proinflammatory cytokines. But the level was greater in NRF2-silenced cells than in HO-1-silenced ones, suggesting the presence of other targets of NRF2. Another stress responsive transcription factor ATF3 is also induced (4.6-fold compared to control [ P  < 0.001]) by NRF2 during infection. Silencing of ATF3 reduced parasite survival (59.3% decrease compared to control [ P  < 0.001]) and increased proinflammatory cytokines. Infection-induced ATF3 recruited HDAC1 into the promoter sites of tumor necrosis factor alpha (TNF-α) and interleukin 12b (IL-12b) genes. Resulting deacetylated histones prevented NF-κB promoter binding, thereby reducing transcription of inflammatory cytokines. Administering the NRF2 inhibitor trigonelline hydrochloride to infected BALB/c mice resulted in reduced HO-1 and ATF3 expression, decreased spleen and liver parasite burdens, and increased proinflammatory cytokine levels. These results suggest that Leishmania upregulates NRF2 to activate both HO-1 and ATF3 for disease progression.

Neutrophils with their array of microbicidal activities are the first innate immune cells to guard against infection. They are also most crucial for the host's initial defense against Leishmania parasites which cause clinically diverse diseases ranging from self-healing cutaneous leishmaniasis (CL) to a more severe visceral form, visceral leishmaniasis (VL). Neutrophils are recruited in large numbers at the infection site after bite of sandfly, which is the vector for the disease. The initial interaction of neutrophils with the parasites may modulate the subsequent innate and adaptive immune responses and hence affect the disease outcome. The purpose of this review is to comprehensively appraise the role of neutrophils during the early stages of Leishmania infection with a focus on the visceral form of the disease. In the past decade, new insights regarding the role of neutrophils in VL have surfaced which have been extensively elaborated in the present review. In addition, since much of the information regarding neutrophil-Leishmania early interaction has accumulated through studies on mouse models of CL, these studies are also revisited. We begin by reviewing the factors which drive the recruitment of neutrophils at the site of injection by the sandfly. We then discuss the studies delineating the molecular mechanisms involved in the uptake of the Leishmania parasite by neutrophils and how the parasite subverts their microbicidal functions. In the end, the interaction of infected neutrophils with macrophages and dendritic cells is summarized., (© 2020 Wiley Periodicals LLC.)

TLR4 activates two distinct signaling pathways involving adaptors MyD88 and TRIF to produce proinflammatory cytokines and type-I interferon respectively. How Leishmania donovani suppresses these pathways is not well studied. We earlier reported, TLR4 is hypersialylated due to reduced membrane-bound neuraminidase (Neu1) on infected-macrophages. We hypothesized that such enhanced sialoglycoconjugates on host cells may modulate the interactions with siglecs- which are the inhibitory receptors. Here, we examined the impact of such sialylation on overall TLR4 activation both in murine cell line J774A.1 and primary bone marrow derived macrophages (BMDM). Supporting this hypothesis, we demonstrated siglec-E engages hypersialylated TLR4 during infection. Such sialic acids-siglec-E interaction enhanced siglec-E phosphorylation that mediated its strong association with SHP1/SHP2 and also upregulated their phosphorylation in both types of macrophages. Pre-treatment of parasites and host cells with neuraminidase reduced SHP1/SHP2 phosphorylation and triggered TLR4 activation respectively through enhanced nuclear translocation of p-65. Moreover, a reciprocal interplay between Neu1 and siglec-E differentially regulates MyD88- and TRIF-pathways through sialic acids on TLR4 as their common substrate during infection. Correspondingly, Neu1 overexpression enhanced MyD88-signaling while still suppressing TRIF-activation. However, silencing siglec-E specifically activated TRIF-signaling. Pro-inflammatory cytokines corresponding to MyD88 and TRIF pathways were also upregulated respectively. Additionally, Neu1 overexpression or siglec-E silencing prevented TLR4 ubiquitination and subsequent degradation by Triad3A. Neu1-overexpression and siglec-E-silencing together followed by infection activated both MyD88 and TRIF-signaling through their enhanced TLR4-association. This elevated the MyD88-specific cytokines and TRIF-mediated IRF3 and IFN-β genes, thus upregulating the pro-inflammatory cytokines and nitric oxide levels and reduced anti-inflammatory cytokines. All these significantly inhibited parasite survival in macrophages thus demonstrating a previously unidentified dualistic regulation of TLR4signaling pathways activation through sialic acids by interplay of Neu1 and siglec-E during Leishmania infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Karmakar and Mandal.)

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease causing high morbidity and mortality in developing countries. Vaccination is considered the most effective and powerful tool for blocking transmission and control of diseases. However, no vaccine is available so far in the market for humans. In the present study, we characterized the hypothetical protein LDBPK&#95;252400 of Leishmania donovani (LdHyP) and explored its prophylactic behavior as a potential vaccine candidate against VL. We found reduced hepato-splenomegaly along with more than 50% parasite reduction in spleen and liver after vaccination in mice. Protection in vaccinated mice after the antigen challenge correlated with the stimulation of antigen specific IFN-γ expressing CD4 + T cell (~4.6 fold) and CD8 + T cells (~2.1 fold) in vaccinated mice in compared to infected mice, even after 2-3 months of immunization. Importantly, antigen-mediated humoral immunity correlated with high antigen specific IgG2/IgG1 responses in vaccinated mice. In vitro re-stimulation of splenocytes with LdHyP enhances the expression of TNF-α, IFN-γ, IL-12 and IL-10 cytokines along with lower IL-4 cytokine and IL-10/IFN-γ ratio in vaccinated mice. Importantly, we observed ~3.5 fold high NO production through activated macrophages validates antigen mediated cellular immunity induction, which is critical in controlling infection progression. These findings suggest that immunization with LdHyP mount a very robust immunity (from IL-10 towards TFN-γ mediated responses) against L. donovani infection and could be explored further as a putative vaccine candidate against VL., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Immunotherapy, a treatment based on host immune system activation, has been shown to provide a substitute for marginally effective conventional chemotherapy in controlling visceral leishmaniasis (VL), the deadliest form of leishmaniasis. As the majority of endemic inhabitants exhibit either subclinical or asymptomatic infection which often develops into the active disease state, therapeutic intervention seems to be an important avenue for combating infections by stimulating the natural defense system of infected individuals. With this perspective, the present study focuses on two immunodominant Leishmania ( L. ) donovani antigens (triosephosphate isomerase and enolase) previously proved to be potent prophylactic VL vaccine candidates, for generating a recombinant chimeric antigen. This is based on the premise that in a heterogeneous population, a multivalent antigen vaccine would be required for an effective response against leishmaniasis (a complex parasitic disease). The resulting molecule rLdT-E chimeric protein was evaluated for its immunogenicity and immunotherapeutic efficacy. A Th1 stimulating adjuvant BCG was employed with the protein which showed a remarkable 70% inhibition of splenic parasitic multiplication positively correlated with boosted Th1 dominant immune response against lethal L. donovani challenge in hamsters as evidenced by high IFN- γ and TNF- α and low IL-10. In addition, immunological analysis of antibody subclass presented IgG2-based humoral response besides considerable delayed-type hypersensitivity and lymphocyte proliferative responses in rLdT-E/BCG-treated animals. Our observations indicate the potential of the chimera towards its candidature for an effective vaccine against Leishmania donovani infection., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Sneha Ratnapriya et al.)

Leishmaniasis is a neglected tropical disease caused by the flagellated protozoa of the genus Leishmania that affects millions of people around the world. Drugs employed in the treatment of leishmaniasis have limited efficacy and induce local and systemic side effects to the patients. Natural products are an interesting alternative to treat leishmaniasis, because some purified molecules are selective toward parasites and not to the host cells. Thus, the aim of the present study was to compare the in vitro antileishmanial activity of the triterpenes betulin (Be), lupeol (Lu), and ursolic acid (UA); analyze the physiology and morphology of affected organelles; analyze the toxicity of selected triterpenes in golden hamsters; and study the therapeutic activity of triterpenes in hamsters infected with L. ( L. ) infantum as well as the cellular immunity induced by studied molecules. The triterpenes Lu and UA were active on promastigote (IC 50 = 4.0 ± 0.3 and 8.0 ± 0.2  μ M, respectively) and amastigote forms (IC 50 = 17.5 ± 0.4 and 3.0 ± 0.2  μ M, respectively) of L. ( L. ) infantum , and their selectivity indexes (SI) toward amastigote forms were higher (≥13.4 and 14, respectively) than SI of miltefosine (2.7). L. ( L. ) infantum promastigotes treated with Lu and UA showed cytoplasmic degradation, and in some of these areas, cell debris were identified, resembling autophagic vacuoles, and parasite mitochondria were swelled, fragmented, and displayed membrane potential altered over time. Parasite cell membrane was not affected by studied triterpenes. Studies of toxicity in golden hamster showed that Lu did not alter blood biochemical parameters associated with liver and kidney functions; however, a slight increase of aspartate aminotransferase level in animals treated with 2.5 mg/kg of UA was detected. Lu and UA triterpenes eliminated amastigote forms in the spleen (87.5 and 95.9% of reduction, respectively) and liver of infected hamster (95.9 and 99.7% of reduction, respectively); and UA showed similar activity at eliminating amastigote forms in the spleen and liver than amphotericin B (99.2 and 99.8% of reduction). The therapeutic activity of both triterpenes was associated with the elevation of IFN- γ and/or iNOS expression in infected treated animals. This is the first comparative work showing the in vitro activity, toxicity, and therapeutic activity of Lu and UA in the chronic model of visceral leishmaniasis caused by L. ( L. ) infantum ; additionally, both triterpenes activated cellular immune response in the hamster model of visceral leishmaniasis., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2021 Jéssica Adriana de Jesus et al.)

Present treatment regimen on visceral leishmaniasis has multiple limitations including severe side effects, toxicity, and resistance of Leishmania strains. Amphotericin B is a well-established pharmacologically approved drug; however, mainly toxicity is a foremost issue with that drug. Recently, our group identified eugenol oleate as an anti-leishmanial immunomodulatory compound. The important objectives of this present study was to evaluate the possible synergistic effect of eugenol oleate with amphotericin B to reduce the toxicity of this approved drug. Results obtained from this study signified that combination of eugenol oleate and amphotericin B showed indifferent combinatorial effect against promastigotes with xΣFIC 1.015, while, moderate synergistic activity with xΣFIC 0.456 against amastigotes. It was also notable that eugenol oleate (2.5 μM) with low concentrations of amphotericin B (0.3125 μM) showed 96.45% parasite reduction within L. donovani-infected murine macrophages. Furthermore, eugenol oleate and amphotericin B significantly (p < 0.01) enhanced the nitrite generation, and pro-inflammatory cytokines (IL-12, IFN-γ and TNF-α) in infected macrophages in vitro and in BALB/c mice in vivo. Eugenol oleate (10 mg/Kg b. wt.) with amphotericin B (1 mg/Kg b.wt.) significantly (p < 0.01) controlled the parasite burden in liver by 96.2% and in spleen by 93.12%. Hence, this study strongly suggested the synergic potential of eugenol oleate with low concentration of amphotericin B in experimental visceral leishmaniasis through anti-leishmanial immune response., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Inflammation is critical for controlling pathogens, but also responsible for symptoms of infectious diseases. IL-27 is an important regulator of inflammation and can limit development of IFNγ-producing Tbet+ CD4+ T (Th1) cells. IL-27 is thought to do this by stimulating IL-10 production by CD4+ T cells, but the underlying mechanisms of these immunoregulatory pathways are not clear. Here we studied the role of IL-27 signalling in experimental visceral leishmaniasis (VL) caused by infection of C57BL/6 mice with the human pathogen Leishmania donovani. We found IL-27 signalling was critical for the development of IL-10-producing Th1 (Tr1) cells during infection. Furthermore, in the absence of IL-27 signalling, there was improved control of parasite growth, but accelerated splenic pathology characterised by the loss of marginal zone macrophages. Critically, we discovered that IL-27 signalling limited glycolysis in Th1 cells during infection that in turn attenuated inflammation. Furthermore, the modulation of glycolysis in the absence of IL-27 signalling restricted tissue pathology without compromising anti-parasitic immunity. Together, these findings identify a novel mechanism by which IL-27 mediates immune regulation during disease by regulating cellular metabolism., Competing Interests: The authors have declared that no competing interests exist.

Leishmania infantum causes visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, which is lethal if untreated. A few years ago, the re-sequencing and de novo assembling of the L. infantum (JPCM5 strain) genome was accomplished, and now we aimed to describe and characterize the experimental proteome of this species. In this work, we performed a proteomic analysis from axenic cultured promastigotes and carried out a detailed comparison with other Leishmania experimental proteomes published to date. We identified 2352 proteins based on a search of mass spectrometry data against a database built from the six-frame translated genome sequence of L. infantum . We detected many proteins belonging to organelles such as glycosomes, mitochondria, or flagellum, as well as many metabolic enzymes and many putative RNA binding proteins and molecular chaperones. Moreover, we listed some proteins presenting post-translational modifications, such as phosphorylations, acetylations, and methylations. On the other hand, the identification of peptides mapping to genomic regions previously annotated as non-coding allowed for the correction of annotations, leading to the N-terminal extension of protein sequences and the uncovering of eight novel protein-coding genes. The alliance of proteomics, genomics, and transcriptomics has resulted in a powerful combination for improving the annotation of the L. infantum reference genome.

The parasitophorous vacuoles (PVs) that insulate Leishmania spp. in host macrophages are vacuolar compartments wherein promastigote forms differentiate into amastigote that are the replicative form of the parasite and are also more resistant to host responses. We revisited the biogenesis of tight-fitting PVs that insulate L. infantum in promastigote-infected macrophage-like RAW 264.7 cells by time-dependent confocal laser multidimensional imaging analysis. Pharmacological disassembly of the cellular microtubule network and silencing of the dynein gene led to an impaired interaction of L. infantum-containing phagosomes with late endosomes and lysosomes, resulting in the tight-fitting parasite-containing phagosomes never transforming into mature PVs. Analysis of the shape of the L. infantum parasite within PVs, showed that factors that impair promastigote-amastigote differentiation can also result in PVs whose maturation is arrested. These findings highlight the importance of the MT-dependent interaction of L. infantum-containing phagosomes with the host macrophage endolysosomal pathway to secure the intracellular fate of the parasite., Competing Interests: The authors have declared that no competing interests exist.

The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified Leishmania donovani heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Enrichment of LdHSP78 in infected humans, hamsters, and parasite amastigotes suggested its importance for disease persistence. Heterozygous knockouts of L. donovani HSP78 ( LdHSP78 +/-) and Leishmania mexicana HSP78 ( LmxHSP78 +/-) were generated using a flanking UTR-based multifragment ligation strategy and the CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. The LdHSP78 +/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, in association with enrichment of proinflammatory cytokines and NO. This finding implies that LdHSP78 +/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Furthermore, phosphorylation of the mitogen-activated protein kinase p38 was enhanced and phosphorylation of extracellular signal-regulated kinase 1/2 was decreased in cells infected with LdHSP78 +/- parasites, compared with WT parasites. Virulence of the LdHSP78 +/- strain was restored by episomal addition of the LdHSP78 gene. Finally, using high-throughput virtual screening, we identified P 1 , P 5 -di(adenosine-5')-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. In summary, using both a genetic knockout approach and pharmacological inhibition, we establish LdHSP78 as a drug target and Ap5A as a potential lead for improved antileishmanial agents., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Das et al.)

Type I interferons (IFNs) induced by an endogenous Leishmania RNA virus or exogenous viral infections have been shown to exacerbate infections with New World Cutaneous Leishmania parasites, however, the impact of type I IFNs in visceral Leishmania infections and implicated mechanisms remain to be unraveled. This study assessed the impact of type I IFN on macrophage infection with L. infantum and L. donovani and the implication of sialoadhesin (Siglec-1/CD169, Sn) as an IFN-inducible surface receptor. Stimulation of bone marrow-derived macrophages with type I IFN (IFN-α) significantly enhanced susceptibility to infection of reference laboratory strains and a set of recent clinical isolates. IFN-α particularly enhanced promastigote uptake. Enhanced macrophage susceptibility was linked to upregulated Sn surface expression as a major contributing factor to the infection exacerbating effect of IFN-α. Stimulation experiments in Sn-deficient macrophages, macrophage pretreatment with a monoclonal anti-Sn antibody or a novel bivalent anti-Sn nanobody and blocking of parasites with soluble Sn restored normal susceptibility levels. Infection of Sn-deficient mice with bioluminescent L. infantum promastigotes revealed a moderate, strain-dependent role for Sn during visceral infection under the used experimental conditions. These data indicate that IFN-responsive Sn expression can enhance the susceptibility of macrophages to infection with visceral Leishmania promastigotes and that targeting of Sn may have some protective effects in early infection., (Copyright © 2020 Van Bockstal, Bulté, Van den Kerkhof, Dirkx, Mabille, Hendrickx, Delputte, Maes and Caljon.)

The protozoan parasite Leishmania donovani (L. donovani) causes visceral leishmaniasis, a chronic infection which is fatal when untreated. Herein, we investigated whether in addition to altering transcription, L. donovani modulates host mRNA translation to establish a successful infection. Polysome-profiling revealed that one third of protein-coding mRNAs expressed in primary mouse macrophages are differentially translated upon infection with L. donovani promastigotes or amastigotes. Gene ontology analysis identified key biological processes enriched for translationally regulated mRNAs and were predicted to be either activated (e.g. chromatin remodeling and RNA metabolism) or inhibited (e.g. intracellular trafficking and antigen presentation) upon infection. Mechanistic in silico and biochemical analyses showed selective activation mTOR- and eIF4A-dependent mRNA translation, including transcripts encoding central regulators of mRNA turnover and inflammation (i.e. PABPC1, EIF2AK2, and TGF-β). L. donovani survival within macrophages was favored under mTOR inhibition but was dampened by pharmacological blockade of eIF4A. Overall, this study uncovers a vast yet selective reprogramming of the host cell translational landscape early during L. donovani infection, and suggests that some of these changes are involved in host defense mechanisms while others are part of parasite-driven survival strategies. Further in vitro and in vivo investigation will shed light on the contribution of mTOR- and eIF4A-dependent translational programs to the outcome of visceral leishmaniasis., Competing Interests: The authors have declared that no competing interests exist.

The major issues in available therapeutic modalities against leishmaniasis are cost, toxicity, and the emergence of drug resistance. The aim of this work was to develop a successful therapeutic adjuvant against drug-resistant Leishmania donovani infection by means of combining Mycobacterium indicus pranii with heat-induced promastigotes (HIP). One-month postinfected BALB/c mice were administered subcutaneously with M. indicus pranii (10 8 cells) and HIP (100 μg) for 5 days. Spleens were harvested for flow cytometric and reverse transcriptase PCR analysis. The antileishmanial effect of the combination strategy was associated with induction of a disease-resolving Th1 and Th17 response with simultaneous downregulation of CD4 + CD25 + Foxp3 + (nTreg) cells and CD4 + CD25 - Foxp3 - (Tr1) cells in the spleen. The significant expansion of CD4 + T CM (CD4 + CD44 hi CD11a hi CD62L hi ) cells was a further interesting outcome of this therapeutic strategy in the context of long-term protection of hosts against secondary infection. Toll-like receptor 2 (TLR2) was also found instrumental in this antiparasitic therapy. Induced interleukin-6 (IL-6) production from expanded CD11c + CD8α + (cDC1) and CD11c + CD11b + (cDC2) dendritic cells (DCs) but not from the CD11b + Ly6c + inflammatory monocytes (iMOs), was found critical in the protective expansion of Th17 as evidenced by an in vivo IL-6 neutralization assay. It also promoted the hematopoietic conversion toward DC progenitors (pre-DCs) from common dendritic cell progenitors (CDPs), the immediate precursors, in bone marrow. This novel combinational strategy demonstrated that expansion of Th17 by IL-6 released from CD11c + classical DCs is crucial, together with the conventional Th1 response, to control drug-resistant infection., (Copyright © 2020 American Society for Microbiology.)

Background: Visceral leishmaniasis/HIV-co-infected patients (VL/HIV) accounts for around 8% of VL reported cases in Brazil. Relapses of Leishmania infection after anti-leishmanial treatment constitute a great challenge in the clinical practice because of the disease severity and drug resistance. We have shown that non-relapsing-VL/HIV (NR-) evolved with increase of CD4 + T-cell counts and reduction of activated CD4 + and CD8 + T cells after anti-leishmanial treatment. This immune profile was not observed in relapsing-VL/HIV patients (R-), indicating a more severe immunological compromising degree. Elevated activation status may be related to a deficient immune reconstitution and could help to explain the frequent relapses in VL/HIV co-infection. Our aim was to evaluate if this gain of T cells was related to changes in the peripheral TCRVβ repertoire and inflammatory status, as well as the possible thymus involvement in the replenishment of these newly formed T lymphocytes. Methods: VL/HIV patients, grouped into non-relapsing (NR- = 6) and relapsing (R- = 12) were evaluated from the active phase up to 12 months post-treatment (mpt). HIV-infected patients (non-VL) and healthy subjects (HS) were included. The TCRVβ repertoire was evaluated ex vivo by flow cytometry, whereas the plasmatic cytokine levels were assessed by Luminex assay. To evaluate the thymic output, DNA was extracted from PBMCs for TCR rearrangement excision circles (TREC) quantification by qPCR. Results: VL/HIV cases presented an altered mobilization profile (expansions or retractions) of the TCRVβ families when compared to HS independent of the follow-up phase ( p < 0.05). TCRVβ repertoire on CD4 + T-cells was more homogeneous in the NR-VL/HIV cases, but heterogeneous on CD8 + T-cells, since different Vβ-families were mobilized. NR-VL/HIV had the inflammatory pattern reduced after 6 mpt. Importantly, VL/HIV patients showed number of TREC copies lower than controls during all follow-up. An increase of recent thymic emigrants was observed in NR-VL/HIV individuals at 10 mpt compared to R- patients ( p < 0.01), who maintained lower TREC contents than the HIV controls. Conclusions: VL/HIV patients that maintain the thymic function, thus generating new T-cells, seem able to replenish the T lymphocyte compartment with effector cells, then enabling parasite control., (Copyright © 2020 Silva-Freitas, Corrêa-Castro, Cota, Giacoia-Gripp, Rabello, Teixeira Dutra, Vasconcelos, Savino, Da-Cruz and Santos-Oliveira.)

The lacuna in the knowledge of immunobiology, especially in visceral infections that are fatal if left untreated, are a major hurdle in getting a vaccine candidate for leishmaniasis. Till date, only a few drugs are available to combat human leishmaniasis and a vaccine candidate either prophylactic or preventive is still awaited. Therefore, identification of host and parasitic factors involved in the regulation of specific immune mechanisms are essentially needed. In this study, we observed that CD200-CD200R immune inhibitory axis regulates host macrophages effectors properties and helps antigen experienced T cells (CD4 + CD44 + T cells) to acquire anti-inflammatory cytokines (IL-4, IL-10, TGF-β, IL-27) producing abilities in an NFkB independent manner. After CD200 blocking the macrophages effectively inhibited proliferation of Leishmania amastigotes and also induced the production of IL-12, IFN-γ, TNF-α and nitric oxide (NOx). Further, the blocking of CD200 signaling also restored macrophages MHC-II expression and helped CD4 + CD44 + T cells to produce pro-inflammatory cytokines like IL-2, IL-12 and IFN-γ. The finding of this study suggested the importance of immune inhibitory mechanisms in controlling Leishmania growth and survival and therefore, requires more studies to understand its role in vaccine induced immunity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Infection with L. donovani affects mainly visceral organs. Importantly, the parasitic load differs in different visceral organs; therefore there is a need to understand the organ specific immune regulation, particularly in the spleen and liver. Comparative studies between these organs in Leishmania infected hamster (Mesocricetus auratus) are lacking. Our study highlights the importance of eicosanoids in the organ specific pathology of visceral leishmaniasis. Among other immune cells, macrophages (mφ) which harbor Leishmania parasite are major producers of eicosanoids. In this study, we intend to explore linkage between organ specific immune response and eicosanoids. We suggest that eicosanoids (early immune modulators) and their organ specific expressions, possibly tune the outcome of mφ differently at different sites. We have observed that liver showed better containment of parasitic load than spleen, where we have found higher expression of 5-lipoxygenase (5-LO) enzyme along with IL-12 and iNOS. However, in spleen, enzymes of the PGE2 pathway i.e. PGE2 synthases (cytosolic and microsomal) along with IL-10 were predominantly higher. To further corroborate our findings, in vitro assays were carried out using purified eicosanoids (LTB4 and PGE2) and the inhibitors of these pathways. Findings establish that the 5-lipoxygenase pathway (i.e. LTB4) is anti-parasitic and its inhibition increases the parasitic load (qPCR based kDNA detection). On the contrary, PGES pathway (i.e. PGE2) supports establishment of infection in mφ. Taken together, 5-LO pathway plays a protective role in liver during L. donovani infection. However, the PGES pathway favors the parasite growth, particularly in the spleen at a later stage., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hypoxia-inducible factor-1 alpha (HIF-1α) is considered a global regulator of cellular metabolism and innate immune cell functions. Intracellular pathogens such as Leishmania have been reported to manipulate host cell metabolism. Herein, we demonstrate that myeloid cells from myeloid-restricted HIF-1α-deficient mice and individuals with loss-of-function HIF1A gene polymorphisms are more susceptible to L. donovani infection through increased lipogenesis. Absence of HIF-1α leads to a defect in BNIP3 expression, resulting in the activation of mTOR and nuclear translocation of SREBP-1c. We observed the induction of lipogenic gene transcripts, such as FASN, and lipid accumulation in infected HIF-1α -/- macrophages. L. donovani-infected HIF-1α-deficient mice develop hypertriglyceridemia and lipid accumulation in splenic and hepatic myeloid cells. Most importantly, our data demonstrate that manipulating FASN or SREBP-1c using pharmacological inhibitors significantly reduced parasite burden. As such, genetic deficiency of HIF-1α is associated with increased lipid accumulation, which results in impaired host-protective anti-leishmanial functions of myeloid cells., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Visceral leishmaniasis (VL) is a tropical and subtropical disease which is endemic in more than eighty countries around the world. Leishmania infantum is one of the main causative agents of VL disease. Currently, there is no approved-to-market vaccine for VL therapy. In this study, we evaluated cellular and humoral immune responses induced by our newly designed multi-epitope vaccine in BALB/c mice. Four antigenic proteins, including histone H1, sterol 24-c-methyltransferase (SMT), Leishmania-specific hypothetical protein (LiHy), and Leishmania-specific antigenic protein (LSAP) were chosen for the prediction of potential immunodominant epitopes. Moreover, to enhance vaccine immunogenicity, two toll-like receptors 4 (TLR4) agonists, resuscitation-promoting factors of Mycobacterium tuberculosis (RpfE and RpfB), were employed as the built-in adjuvants. Immunization with the designed multi-epitope vaccine elicited a robust Th1-type immune response, compared to other groups, as shown by increased levels of IL-2, IFN-γ, TNF-α, and IgG2a. Furthermore, a significant decrease was observed in Th-2-type-related cytokines such as IL-4 in immunized mice. The designed construct also induced a significant reduction in parasite load (p < 0.0001), conferring protection against L. infantum challenge. This study could be promising in gaining insight towards the potential of peptide epitope-based vaccines as effective protective approaches against Leishmania species.

Myeloid-related protein 14 (MRP14) belongs to the S100 calcium-binding protein family and is expressed in neutrophils and inflammatory macrophages. Increase in the number of MRP14+ cells or serum level of MRP14 is associated with various diseases such as autoimmune diseases and infectious diseases, suggesting the involvement of the molecule in pathogenesis of those diseases. In this study, to examine the pathological involvement of MRP14 during cutaneous and visceral leishmaniasis, wild-type (WT) and MRP14 knockout (MRP14KO) mice were infected with Leishmania major and L. donovani. Increase in the number of MRP14+ cells at the infection sites in wild-type mice was commonly found in the skin during L. major infection as well as the spleen and liver during L. donovani infection. In contrast, the influence of MRP14 to the pathology seemed different between the two infections. MRP14 depletion exacerbated the lesion development and ulcer formation in L. major infection. On the other hand, the depletion improved anemia and splenomegaly but not hepatomegaly at 24 weeks of L. donovani infection. These results suggest that, distinct from its protective role in CL, MRP14 is involved in exacerbation of some symptoms during VL., Competing Interests: The authors have declared that no competing interests exist.

Cyanine compounds have previously shown excellent in vitro and promising in vivo antileishmanial efficacy, but the potential toxicity of these agents is a concern. A series of 22 analogs of thiazole orange ((Z)-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium salt), a commercial cyanine dye with antileishmanial activity, were synthesized in an effort to increase the selectivity of such compounds while maintaining efficacy. Cyanines possessing substitutions on the quinolinium ring system displayed potency against Leishmania donovani axenic amastigotes that differed little from the parent compound (IC 50 12-42 nM), while ring disjunction analogs were both less potent and less toxic. Changes in DNA melting temperature were modest when synthetic oligonucleotides were incubated with selected analogs (ΔTm ≤ 5 °C), with ring disjunction analogs showing the least effect on this parameter. Despite the high antileishmanial potency of the target compounds, their toxicity and relatively flat SAR suggests that further information regarding the target(s) of these molecules is needed to aid their development as antileishmanials., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

β-amino acids and their analogues are gathering increased attention not only because of their antibacterial and antifungal activity, but also for their use in designing peptidomimetics with increased oral bioavailability and resistance to metabolic degradation. In this study, a series of α-phenyl substituted chalcones, α-phenyl, β-amino substituted dihydrochalcones and β-amino acid derivatives were synthesized and evaluated for their antileishmanial efficacy against experimental visceral leishmaniasis (VL). Among all synthesized derivatives, 10c showed promising antileishmanial efficacy against both extracellular promastigote and intracellular amastigote (IC 50 8.2 μM and 20.5 μM respectively) of L. donovani with negligible cytotoxic effect towards J774 macrophages and Vero cells. 10c effectively reduced spleen and liver parasite burden (>90%) in both hamster and Balb/c model of VL without any hepatotoxicity. In vitro pharmacokinetic analysis showed that 10c was stable in gastric fluid and plasma of Balb/c mice at 10 μg/ml. Further analysis of the molecular mechanism revealed that 10c entered into the parasite by depolarizing the plasma membrane rather than forming nonspecific pores and induced molecular events like loss in mitochondrial membrane potential with a gradual decline in ATP production. This, in turn, did not induce programmed cell death of the parasite; rather 10c induced bioenergetic collapse of the parasite by decreasing ATP synthesis through specific inhibition of mitochondrial complex III activity. Altogether, our results allude to the therapeutic potential of β-amino acid derivatives as novel antileishmanials, identifying them as lead compounds for further exploration in the design of potent candidates for the treatment of visceral leishmaniasis., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Visceral leishmaniasis is a deadly illness caused by Leishmania donovani that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of L. major , named inhibitor of serine peptidases (ISP) 2, inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF-α and IFN-β production, and parasite survival. We report poor intracellular growth of L. donovani in macrophages from knockout mice for NE ( ela -/- ), TLR4, or TLR2. NE and TLR4 colocalized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of ela -/- mice were reduced and accompanied by increased NO and decreased TGF-β production. Expression of ISP2 was not detected in L. donovani , and a transgenic line constitutively expressing ISP 2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected ela -/- macrophages displayed significantly lower IFN-β mRNA than background mice macrophages, and the intracellular growth was fully restored by exogenous IFN-β. We propose that L. donovani utilizes the host NE-TLR machinery to induce IFN-β necessary for parasite survival and growth during early infection. Low or absent expression of parasite ISP2 in L. donovani is necessary to preserve the activation of the NE-TLR pathway.-Dias, B. T., Dias-Teixeira, K. L., Godinho, J. P., Faria, M. S., Calegari-Silva, T., Mukhtar, M. M., Lopes, U. G., Mottram, J. C., Lima, A. P. C. A. Neutrophil elastase promotes Leishmania donovani infection via interferon-β.

Protein malnutrition is a risk factor for developing visceral leishmaniasis. Because we previously demonstrated that protein malnutrition and infection with Leishmania infantum disrupts the splenic microarchitecture in BALB/c mice, alters T cell-subsets and increases splenic parasite load, we hypothesize that splenic microenvironment is precociously compromised in infected animals that suffered a preceding malnutrition. To evaluate this, we characterized the abundance of proteins secreted in the splenic interstitial fluid (IF) using an iTRAQ-based quantitative proteomics approach. In addition, local levels of pro-inflammatory and proliferation molecules were analyzed. Whereas well-nourished infected animals showed increased IL-1β and IL-2 levels, malnourished-infected mice displayed significant reduction of these cytokines. Remarkably, a two-weeks infection with L. infantum already modified protein abundance in the splenic IF of well-nourished mice, but malnourished animals failed to respond to infection in the same fashion. Malnutrition induced significant reduction of chemotactic and pro-inflammatory molecules as well as of proteins involved in nucleic acid and amino acid metabolism, indicating an impaired proliferative microenvironment. Accordingly, a significant decrease in Ki67 expression was observed, suggesting that splenocyte proliferation is compromised in malnourished animals. Together, our results show that malnutrition compromises the splenic microenvironment and alters the immune response to the parasite in malnourished individuals. SIGNIFICANCE: Protein malnutrition is recognized as an important epidemiological risk factor for developing visceral leishmaniasis (VL). Locally secreted factors present in the interstitial fluid have important roles in initiating immune responses and in regulating fluid volume during inflammation. However, the regulation of secreted factors under pathological conditions such as malnutrition and infection are widely unknown. To analyze how protein malnutrition alters secreted proteins involved in the immune response to L. infantum infection we evaluated the proteomic profile of the interstitial fluid of the spleen in malnourished BALB/c mice infected with L. infantum. Our work revealed new elements that contribute to the understanding of the immunopathological events in the spleen of malnourished animals infected with L. infantum and opens new pathways for consideration of other aspects that could improve VL treatment in malnourished individuals., (Copyright © 2019 Elsevier B.V. All rights reserved.)