Antimony-Resistant Leishmania donovani Exploits miR-466i To Deactivate Host MyD88 for Regulating IL-10/IL-12 Levels during Early Hours of Infection.

Infection with antimony-resistant Leishmania donovani (Sb(R)LD) induces aggressive pathology in the mammalian hosts as compared with ones with antimony-sensitive L. donovani (Sb(S)LD) infection. Sb(R)LD, but not Sb(S)LD, interacts with TLR2/TLR6 to induce IL-10 by exploiting p50/c-Rel subunits of NF-κB in infected macrophages (Mϕs). Most of the TLRs exploit the universal adaptor protein MyD88 to activate NF-κB. We now show that infection of Mϕs from MyD88(-/-) mice with Sb(R)LD gave rise to significantly higher intracellular parasite number coupled with elevated IL-10/IL-12 ratio in the culture supernatant as compared with infection in wild type (WT) Mϕs. Τhese attributes were not seen with Sb(S)LD in similar experiments. Further, Sb(R)LD infection upregulated miR-466i, which binds with 3'-untranslated region, leading to the downregulation of MyD88. Infection of MyD88(-/-) Mϕ or IL-12(-/-) Mϕ with Sb(R)LD induced IL-10 surge at 4 h, whereas the same in WT Mϕ started from 12 h. Thus, absence of IL-12 in MyD88(-/-) mice favored early binding of NF-κB subunits to the IL-10 promoter, resulting in IL-10 surge. Infection of MyD88(-/-) mice with Sb(R)LD showed significantly higher organ parasites coupled with ill-defined and immature hepatic granulomas, whereas in WT mice there were less organ parasites and the granulomas were well defined. From the survival kinetics it was observed that Sb(R)LD-infected MyD88(-/-) mice died by 60 d postinfection, whereas the WT mice continued to survive. Our results demonstrate that Sb(R)LD has evolved a unique strategy to evade host antileishmanial immune repertoire by manipulating host MyD88 to its advantage.

Liposomal cholesterol delivery activates the macrophage innate immune arm to facilitate intracellular Leishmania donovani killing.

Leishmania donovani causes visceral leishmaniasis (VL) by infecting the monocyte/macrophage lineage and residing inside specialized structures known as parasitophorous vacuoles. The protozoan parasite has adopted several means of escaping the host immune response, with one of the major methods being deactivation of host macrophages. Previous reports highlight dampened macrophage signaling, defective antigen presentation due to increased membrane fluidity, and the downregulation of several genes associated with L. donovani infection. We have reported previously that the defective antigen presentation in infected hamsters could be corrected by a single injection of a cholesterol-containing liposome. Here we show that cholesterol in the form of a liposomal formulation can stimulate the innate immune arm and reactivate macrophage function. Augmented levels of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI), along with proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), corroborate intracellular parasite killing. Cholesterol incorporation kinetics is favored in infected macrophages more than in normal macrophages. Such an enhanced cholesterol uptake is associated with preferential apoptosis of infected macrophages in an endoplasmic reticulum (ER) stress-dependent manner. All these events are coupled with mitogen-activated protein (MAP) kinase activation, while inhibition of such pathways resulted in increased parasite loads. Hence, liposomal cholesterol is a potential facilitator of the macrophage effector function in favor of the host, independently of the T-cell arm.

NAD(P)H cytochrome b5 oxidoreductase deficiency in Leishmania major results in impaired linoleate synthesis followed by increased oxidative stress and cell death.

NAD(P)H cytochrome b(5) oxidoreductase (Ncb5or), comprising cytochrome b(5) and cytochrome b(5) reductase domains, is widely distributed in eukaryotic organisms. Although Ncb5or plays a crucial role in lipid metabolism of mice, so far no Ncb5or gene has been reported in the unicellular parasitic protozoa Leishmania species. We have cloned, expressed, and characterized Ncb5or gene from Leishmania major. Steady state catalysis and spectral studies show that NADH can quickly reduce the ferric state of the enzyme to the ferrous state and is able to donate an electron(s) to external acceptors. To elucidate its exact physiological role in Leishmania, we attempted to create NAD(P)H cytochrome b(5) oxidoreductase from L. major (LmNcb5or) knock-out mutants by targeted gene replacement technique. A free fatty acid profile in knock-out (KO) cells reveals marked deficiency in linoleate and linolenate when compared with wild type (WT) or overexpressing cells. KO culture has a higher percentage of dead cells compared with both WT and overexpressing cells. Increased O(2) uptake, uncoupling and ATP synthesis, and loss of mitochondrial membrane potential are evident in KO cells. Flow cytometric analysis reveals the presence of a higher concentration of intracellular H(2)O(2), indicative of increased oxidative stress in parasites lacking LmNcb5or. Cell death is significantly reduced when the KO cells are pretreated with BSA bound linoleate. Real time PCR studies demonstrate a higher Δ12 desaturase, superoxide dismutase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA with a concomitant fall in Δ9 desaturase mRNA expression in LmNcb5or null cell line. Together these findings suggest that decreased linoleate synthesis, and increased oxidative stress and apoptosis are the major consequences of LmNcb5or deficiency in Leishmania.

Hyperlipidemia offers protection against Leishmania donovani infection: role of membrane cholesterol.

Leishmania donovani (LD), the causative agent of visceral leishmaniasis (VL), extracts membrane cholesterol from macrophages and disrupts lipid rafts, leading to their inability to stimulate T cells. Restoration of membrane cholesterol by liposomal delivery corrects the above defects and offers protection in infected hamsters. To reinforce further the protective role of cholesterol in VL, mice were either provided a high-cholesterol (atherogenic) diet or underwent statin treatment. Subsequent LD infection showed that an atherogenic diet is associated with protection, whereas hypocholesterolemia due to statin treatment confers susceptibility to the infection. This observation was validated in apolipoprotein E knockout mice (AE) mice that displayed intrinsic hypercholesterolemia with hepatic granuloma, production of host-protective cytokines, and expansion of antileishmanial CD8(+)IFN- γ (+) and CD8(+)IFN- γ (+)TNF- α (+) T cells in contrast to the wild-type C57BL/6 (BL/6) mice when infected with LD. Normal macrophages from AE mice (N-AE-MΦ) showed 3-fold higher membrane cholesterol coupled with increased fluorescence anisotropy (FA) compared with wild-type macrophage (N-BL/6-MΦ). Characterization of in vitro LD-infected AE macrophage (LD-AE-MΦ) revealed intact raft architecture and ability to stimulate T cells, which were compromised in LD-BL/6-MΦ. This study clearly indicates that hypercholesterolemia, induced intrinsically or extrinsically, can control the pathogenesis of VL by modulating immune repertoire in favor of the host.

Antimony resistance mechanism in genetically different clinical isolates of Indian Kala-azar patients.

The central theme of this enterprise is to find common features, if any, displayed by genetically different antimony (Sb)-resistant viscerotropic Leishmania parasites to impart Sb resistance. In a limited number of clinical isolates (n = 3), we studied the breadth of variation in the following dimensions: (a) intracellular thiol content, (b) cell surface expression of glycan having N-acetyl-D-galactosaminyl residue as the terminal sugar, and (c) gene expression of thiol-synthesizing enzymes (CBS, MST, gamma-GCS, ODC, and TR), antimony-reducing enzymes (TDR and ACR2), and antimonial transporter genes (AQP1, MRPA, and PRP1). One of the isolates, T5, that was genotypically characterized as Leishmania tropica, caused Indian Kala-azar and was phenotypically Sb resistant (T5-LT-SSG-R), while the other two were Leishmania donovani, out of which one isolate, AG83, is antimony sensitive (AG83-LD-SSG-S) and the other isolate, T8, is Sb resistant (T8-LD-SSG-R). Our study showed that the Sb-resistant parasites, regardless of their genotype, showed significantly higher intracellular thiol compared with Sb-sensitive AG83-LD-SSG-S. Seemingly, T5-LT-SSG-R showed about 1.9-fold higher thiol content compared with T8-LD-SSG-R which essentially mirrored cell surface N-acetyl-D-galactosaminyl expression. Except TR, the expression of the remaining thiol-synthesizing genes was significantly higher in T8-LD-SSG-R and T5-LT-SSG-R than the sensitive one, and between the Sb-resistant parasites, the latter showed a significantly higher expression. Furthermore, the genes for Sb-reducing enzymes increased significantly in resistant parasites regardless of genotype compared with the sensitive one, and between two resistant parasites, there was hardly any difference in expression. Out of three antimony transporters, AQP1 was decreased with the concurrent increase in MRPA and PRP1 in resistant isolates when compared with the sensitive counterpart. Interestingly, no difference in expression of the above-mentioned transporters was noted between two Sb-resistant isolates. The enduring image that resonated from our study is that the genetically diverse Sb-resistant parasites showed enhanced thiol-synthesizing and antimony transporter gene expression than the sensitive counterpart to confer a resistant phenotype.

Modulation of Splenic B Cell Subsets during Experimental Leishmania donovani Infection in BALB/c Mice.

Sodium antimonials are one of the major and common drugs used against visceral form leishmaniasis (VL). However, the development of drug resistance makes it difficult to manage this disease. Current work investigates the modulation of splenic B cells during experimental infection with antimony-sensitive and -resistant Leishmania donovani infection. Here we phenotypically characterized splenic B cell subsets in BALB/c mice infected with antimony drug-sensitive and -resistant VL strains using flow-cytometry method. In the splenocytes we noticed increased number of Transitional T3 B cells and B1a B cells in drug-resistant VL strain infection. Besides, we also observed alteration in Follicular B cell population of antimony-resistant strain infected mice. Drug-resistant strain induced secretion of elevated level of IL-10 from B1a B cells and IL-6 from Transitional T3 B cell subsets in the splenocytes. Purified splenic B cells from antimony drug-resistant strain infected mice showed decrease in the Lyn kinase gene expression compared to sensitive strain infected and uninfected mice. The current study provides insight into changes in host splenic B-cell subsets during experimental infection with antimony-sensitive and -resistant L. donovani in murine model.

Short-Course Treatment With Imipramine Entrapped in Squalene Liposomes Results in Sterile Cure of Experimental Visceral Leishmaniasis Induced by Antimony Resistant Leishmania donovani With Increased Efficacy.

Previously we have shown that long term oral treatment of tricyclic-antidepressant-drug, imipramine, against experimental visceral leishmaniasis, results in clearance of organ parasites, regardless of input infection, either with antimony-sensitive (SbS) or antimony-resistant (SbR) Leishmania donovani (LD) clinical isolates. Although continuous imipramine monotherapy for 28 days (5 mg/kg) results in significant clearance of organ parasites in both SbR and SbSLD infected hamsters, the dose for the sterile parasite clearance from visceral organ is comparatively higher (10 mg/kg) and shows signs of toxicity. Hence, to reduce the toxicity, we encapsulated imipramine in squalene-phosphatidylcholine (SP) liposome (Lip-Imi) and tested its efficacy for a short-course treatment (10 days) in the animal model of visceral leishmaniasis. We observed a significant reduction of hepatic toxicity coupled with sterile parasite clearance in case of this short-course treatment of Lip-Imi, which is absent with free Imi treatment. This also correlates with significant increase in serum availability of imipramine in case of Lip-Imi treatment due to sustained release. Clearance of parasite was coupled with the polarization of antileishmanial immune repertoire from Th2 to Th1 after treatment with Lip-Imi in both SbRLD and SbSLD infected mouse models of LD infection. This study showed that imipramine is effective against both SbSLD and SbRLD at a significantly lower dose with reduced time course of treatment without any toxic side effects, when encapsulated in SP-liposome. Thus, the drug has the potential to be repurposed for the treatment of Kala-azar.

Genetic markers for antimony resistant clinical isolates differentiation from Indian Kala-azar.

Visceral Leishmaniasis or Kala-azar is caused by the protozoan parasites belonging to the Genus Leishmania. Once thought eradicated from the Indian subcontinent, the disease came back with drug resistance to almost all prevalent drugs. Molecular epidemiological studies revealed the polymorphic nature of the population of the main player of the disease, Leishmania donovani and involvement of other species (L. tropica) and other genus (Leptomonas) with the disease. This makes control measures almost futile. It also strongly demands the characterization of each and every isolate mandatory which is not done. In this background, the present study has been carried out to assess the genetic attributes of each clinical isolates (n=26) of KA and PKDL patients from India and Bangladesh. All the isolates were characterized through Restriction Fragment Length Polymorphism (RFLP) analysis to ascertain their species identity. 46.2% of the isolates were found to be Sodium Stibogluconate (SSG) resistant by amastigote-macrophage model. When the clinical isolates were subjected to Single Stranded Conformation Polymorphism (SSCP) of Internal Transcribed Spacer 1 (ITS1), Internal Transcribed Spacer 2 (ITS2) and some anonymous markers, the drug resistant Leishmania isolates of SSG can be distinguished from the sensitive isolates distinctly. This study showed for the first time, the genetic markers for SSG drug resistance of Indian Kala-azar clinical isolates.

Antimony resistant Leishmania donovani but not sensitive ones drives greater frequency of potent T-regulatory cells upon interaction with human PBMCs: role of IL-10 and TGF-ß in early immune response.

In India the sand fly, Phlebotomus argentipes, transmitted parasitic disease termed kala-azar is caused by Leishmania donovani (LD) in humans. These immune-evading parasites have increasingly developed resistance to the drug sodium antimony gluconate in endemic regions. Lack of early diagnosis methods for the disease limits the information available regarding the early interactions of this parasite with either human tissues or cell lineages. We reasoned that peripheral blood mononuclear cells (PBMCs) from healthy human beings could help compare some of their immune signatures once they were exposed for up to 8 days, to either pentavalent antimony sensitive (Sb(S)-LD) or resistant (Sb(R)-LD) Leishmania donovani isolates. At day 2, PBMC cultures exposed to Sb(S)-LD and Sb(R)-LD stationary phase promastigotes had four and seven fold higher frequency of IL-10 secreting monocyte-macrophage respectively, compared to cultures unexposed to parasites. Contrasting with the CD4(+)CD25-CD127- type-1 T-regulatory (Tr1) cell population that displayed similar features whatever the culture conditions, there was a pronounced increase in the IL-10 producing CD4(+)CD25(+)CD127low/- inducible T-regulatory cells (iTregs) in the PBMC cultures sampled at day 8 post addition of Sb(R)-LD. Sorted iTregs from different cultures on day 8 were added to anti-CD3/CD28 induced naïve PBMCs to assess their suppressive ability. We observed that iTregs from Sb(R)-LD exposed PBMCs had more pronounced suppressive ability compared to Sb(S)-LD counterpart on a per cell basis and is dependent on both IL-10 and TGF-ß, whereas IL-10 being the major factor contributing to the suppressive ability of iTregs sorted from PBMC cultures exposed to Sb(S)-LD. Of note, iTreg population frequency value remained at the basal level after addition of genetically modified Sb(R)-LD lacking unique terminal sugar in surface glycan. Even with limitations of this artificial in vitro model of L. donovani-human PBMC interactions, the present findings suggest that Sb(R)-LD have higher immunomodulatory capacity which may favour aggressive pathology.

Modular multiantigen T cell epitope-enriched DNA vaccine against human leishmaniasis.

The leishmaniases are protozoal diseases that severely affect large populations in tropical and subtropical regions. There are only limited treatment options and preventative measures. Vaccines will be important for prevention, control and elimination of leishmaniasis, and could reduce the transmission and burden of disease in endemic populations. We report the development of a DNA vaccine against leishmaniasis that induced T cell-based immunity and is a candidate for clinical trials. The vaccine antigens were selected as conserved in various Leishmania species, different endemic regions, and over time. They were tested with T cells from individuals cured of leishmaniasis, and shown to be immunogenic and to induce CD4(+) and CD8(+) T cell responses in genetically diverse human populations of different endemic regions. The vaccine proved protective in a rodent model of infection. Thus, the immunogenicity of candidate vaccine antigens in human populations of endemic regions, as well as proof of principle for induction of specific immune responses and protection against Leishmania infection in mice, provides a viable strategy for T cell vaccine development.

Heterologous priming-boosting with DNA and vaccinia virus expressing kinetoplastid membrane protein-11 induces potent cellular immune response and confers protection against infection with antimony resistant and sensitive strains of Leishmania (Leishmania) donovani.

BACKGROUND: Emergence of resistance against commonly available drugs poses a major threat in the treatment of visceral leishmaniasis (VL), particularly in the Indian subcontinent. Absence of any licensed vaccine against VL emphasizes the urgent need to develop an effective alternative vaccination strategy. METHODOLOGY: We developed a novel heterologous prime boost immunization strategy using kinetoplastid membrane protein-11 (KMP-11) DNA priming followed by boosting with recombinant vaccinia virus (rVV) expressing the same antigen. The efficacy of this vaccination regimen in a murine and hamster model of visceral leishmaniasis caused by both antimony resistant (Sb-R) and sensitive (Sb-S) Leishmania (L.) donovani is examined. RESULT: Heterologous prime-boost (KMP-11 DNA/rVV) vaccination was able to protect mice and hamsters from experimental VL induced by both Sb-S and Sb-R-L. (L.) donovani isolates. Parasite burden is kept significantly low in the vaccinated groups even after 60 days post-infection in hamsters, which are extremely susceptible to VL. Protection in mice is correlated with strong cellular and humoral immune responses. Generation of polyfunctional CD8(+) T cell was observed in vaccinated groups, which is one of the most important prerequisite for successful vaccination against VL. Protection was accompanied with generation of antigen specific CD4(+) and CD8(+) cells that produced effector cytokines such as IFN-γ, IL-2 and TNF-α. KMP-11-DNA/rVV vaccination also developed strong cytotoxic response and reversed T-cell impairment to induce antigen specific T cell proliferation. CONCLUSION: KMP-11 is a unique antigen with high epitope density. Heterologous prime boost vaccination activates CD4(+) and CD8(+) T-cell mediated immunity to confer resistance to VL. This immunization method also produces high quality T-cells secreting multiple effector cytokines thus enhancing durability of the immune response. Thus the vaccination regime as described in the present study could provide a potent strategy for future anti-leishmanial vaccine development.