Guanylate Binding Proteins Restrict Leishmania donovani Growth in Nonphagocytic Cells Independent of Parasitophorous Vacuolar Targeting.

Interferon (IFN)-inducible guanylate binding proteins (GBPs) play important roles in host defense against many intracellular pathogens that reside within pathogen-containing vacuoles (PVs). For instance, members of the GBP family translocate to PVs occupied by the protozoan pathogen Toxoplasma and facilitate PV disruption and lytic parasite killing. While the GBP defense program targeting Toxoplasma has been studied in some detail, the role of GBPs in host defense to other protozoan pathogens is poorly characterized. Here, we report a critical role for both mouse and human GBPs in the cell-autonomous immune response against the vector-borne parasite Leishmania donovani Although L. donovani can infect both phagocytic and nonphagocytic cells, it predominantly replicates inside professional phagocytes. The underlying basis for this cell type tropism is unclear. Here, we demonstrate that GBPs restrict growth of L. donovani in both mouse and human nonphagocytic cells. GBP-mediated restriction of L. donovani replication occurs via a noncanonical pathway that operates independent of detectable translocation of GBPs to L. donovan-containing vacuoles (LCVs). Instead of promoting the lytic destruction of PVs, as reported for GBP-mediated killing of Toxoplasma in phagocytic cells, GBPs facilitate the delivery of L. donovani into autolysosomal-marker-positive compartments in mouse embryonic fibroblasts as well as the human epithelial cell line A549. Together our results show that GBPs control a novel cell-autonomous host defense program, which renders nonphagocytic cells nonpermissible for efficient Leishmania replication.IMPORTANCE The obligate intracellular parasite Leishmania causes the disease leishmaniasis, which is transmitted to mammalian hosts, including humans, via the sandfly vector. Following the bite-induced breach of the skin barrier, Leishmania is known to live and replicate predominantly inside professional phagocytes. Although Leishmania is also able to infect nonphagocytic cells, nonphagocytic cells support limited parasitic replication for unknown reasons. In this study, we show that nonphagocytic cells possess an intrinsic property to restrict Leishmania growth. Our study defines a novel role for a family of host defense proteins, the guanylate binding proteins (GBPs), in antileishmanial immunity. Mechanistically, our data indicate that GBPs facilitate the delivery of Leishmania into antimicrobial autolysosomes, thereby enhancing parasite clearance in nonphagocytic cells. We propose that this GBP-dependent host defense program makes nonphagocytic cells an inhospitable host cell type for Leishmania growth.

Tryptophan-kynurenine pathway attenuates ß-catenin-dependent pro-parasitic role of STING-TICAM2-IRF3-IDO1 signalosome in Toxoplasma gondii infection.

Recent studies have documented the diverse role of host immunity in infection by the protozoan parasite, Toxoplasma gondii. However, the contribution of the ß-catenin pathway in this process has not been explored. Here, we show that AKT-mediated phosphorylated ß-catenin supports T. gondii multiplication which is arrested in the deficiency of its phosphorylation domain at S552 position. The ß-catenin-TCF4 protein complex binds to the promoter region of IRF3 gene and initiates its transcription, which was also abrogated in ß-catenin knockout cells. TBK-independent phosphorylation of STING(S366) and its adaptor molecule TICAM2 by phospho-AKT(T308S473) augmented downstream IRF3-dependent IDO1 transcription, which was also dependent on ß-catenin. But, proteasomal degradation of IDO1 by its tyrosine phosphorylation (at Y115 and Y253) favoured parasite replication. In absence of IDO1, tryptophan was catabolized into melatonin, which supressed cellular reactive oxygen species (ROS) and boosted parasite growth. Conversely, when tyrosine phosphorylation was abolished by phosphosite mutations, IDO1 escaped its ubiquitin-mediated proteasomal degradation system (UPS) and the stable IDO1 prevented parasite replication by kynurenine synthesis. We propose that T. gondii selectively utilizes tryptophan to produce the antioxidant, melatonin, thus prolonging the survival of infected cells through functional AKT and ß-catenin activity for better parasite replication. Stable IDO1 in the presence of IFN-γ catabolized tryptophan into kynurenine, promoting cell death by suppressing phospho-AKT and phospho-ß-catenin levels, and circumvented parasite replication. Treatment of infected cells with kynurenine or its analogue, teriflunomide suppressed kinase activity of AKT, and phosphorylation of ß-catenin triggering caspase-3 dependent apoptosis of infected cells to inhibit parasite growth. Our results demonstrate that ß-catenin regulate phosphorylated STING-TICAM2-IRF3-IDO1 signalosome for a cell-intrinsic pro-parasitic role. We propose that the downstream IRF3-IDO1-reliant tryptophan catabolites and their analogues can act as effective immunotherapeutic molecules to control T. gondii replication by impairing the AKT and ß-catenin axis.

Radio-attenuated leishmanial parasites as immunoprophylactic agent against experimental murine visceral leishmaniasis.

The present study intends to evaluate the role of radio-attenuated leishmania parasites as immunoprophylactic agents for experimental murine visceral leishmaniasis. BALB/c mice were immunized with gamma (γ)-irradiated Leishmania donovani. A second immunization was given after 15 days of first immunization. After two immunizations, mice were infected with virulent L. donovani promastigotes. Protection against Kala-azar (KA) was estimated from spleen and liver parasitic burden along with the measurement of nitrite and superoxide anion generation by isolation of splenocytes and also by T-lymphocyte helper 1(Th1) and T-lymphocyte helper 2(Th2) cytokines release from the experimental groups. It was observed that BALB/c mice having prior immunization with radio-attenuated parasites showed protection against L. donovani infection through higher expression of Th1 cytokines and suppression of Th2 cytokines along with the generation of protective free radicals. The group of mice without prior priming with radio-attenuated parasites surrendered to the disease. Thus it can be concluded that radio-attenuated L. donovani may be used for.

Use of antimony in the treatment of leishmaniasis: current status and future directions.

In the recent past the standard treatment of kala-azar involved the use of pentavalent antimonials Sb(V). Because of progressive rise in treatment failure to Sb(V) was limited its use in the treatment program in the Indian subcontinent. Until now the mechanism of action of Sb(V) is not very clear. Recent studies indicated that both parasite and hosts contribute to the antimony efflux mechanism. Interestingly, antimonials show strong immunostimulatory abilities as evident from the upregulation of transplantation antigens and enhanced T cell stimulating ability of normal antigen presenting cells when treated with Sb(V) in vitro. Recently, it has been shown that some of the peroxovanadium compounds have Sb(V)-resistance modifying ability in experimental infection with Sb(V) resistant Leishmania donovani isolates in murine model. Thus, vanadium compounds may be used in combination with Sb(V) in the treatment of Sb(V) resistance cases of kala-azar.

Leishmania donovani isolates with antimony-resistant but not -sensitive phenotype inhibit sodium antimony gluconate-induced dendritic cell activation.

The inability of sodium antimony gluconate (SAG)-unresponsive kala-azar patients to clear Leishmania donovani (LD) infection despite SAG therapy is partly due to an ill-defined immune-dysfunction. Since dendritic cells (DCs) typically initiate anti-leishmanial immunity, a role for DCs in aberrant LD clearance was investigated. Accordingly, regulation of SAG-induced activation of murine DCs following infection with LD isolates exhibiting two distinct phenotypes such as antimony-resistant (Sb(R)LD) and antimony-sensitive (Sb(S)LD) was compared in vitro. Unlike Sb(S)LD, infection of DCs with Sb(R)LD induced more IL-10 production and inhibited SAG-induced secretion of proinflammatory cytokines, up-regulation of co-stimulatory molecules and leishmanicidal effects. Sb(R)LD inhibited these effects of SAG by blocking activation of PI3K/AKT and NF-kappaB pathways. In contrast, Sb(S)LD failed to block activation of SAG (20 microg/ml)-induced PI3K/AKT pathway; which continued to stimulate NF-kappaB signaling, induce leishmanicidal effects and promote DC activation. Notably, prolonged incubation of DCs with Sb(S)LD also inhibited SAG (20 microg/ml)-induced activation of PI3K/AKT and NF-kappaB pathways and leishmanicidal effects, which was restored by increasing the dose of SAG to 40 microg/ml. In contrast, Sb(R)LD inhibited these SAG-induced events regardless of duration of DC exposure to Sb(R)LD or dose of SAG. Interestingly, the inhibitory effects of isogenic Sb(S)LD expressing ATP-binding cassette (ABC) transporter MRPA on SAG-induced leishmanicidal effects mimicked that of Sb(R)LD to some extent, although antimony resistance in clinical LD isolates is known to be multifactorial. Furthermore, NF-kappaB was found to transcriptionally regulate expression of murine gammaglutamylcysteine synthetase heavy-chain (mgammaGCS(hc)) gene, presumably an important regulator of antimony resistance. Importantly, Sb(R)LD but not Sb(S)LD blocked SAG-induced mgammaGCS expression in DCs by preventing NF-kappaB binding to the mgammaGCS(hc) promoter. Our findings demonstrate that Sb(R)LD but not Sb(S)LD prevents SAG-induced DC activation by suppressing a PI3K-dependent NF-kappaB pathway and provide the evidence for differential host-pathogen interaction mediated by Sb(R)LD and Sb(S)LD.

Sub-optimal dose of Sodium Antimony Gluconate (SAG)-diperoxovanadate combination clears organ parasites from BALB/c mice infected with antimony resistant Leishmania donovani by expanding antileishmanial T-cell repertoire and increasing IFN-gamma to IL-10 ratio.

We demonstrate that the combination of sub-optimal doses of Sodium Antimony Gluconate (SAG) and the diperoxovanadate compound K[VO(O2)2(H2O)], also designated as PV6, is highly effective in combating experimental infection of BALB/c mice with antimony resistant (Sb(R)) Leishmania donovani (LD) as evident from the significant reduction in organ parasite burden where SAG is essentially ineffective. Interestingly, such treatment also allowed clonal expansion of antileishmanial T-cells coupled with robust surge of IFN-c and concomitant decrease in IL-10 production. The splenocytes from the treated animals generated significantly higher amounts of IFN-c inducible parasiticidal effector molecules like superoxide and nitric oxide as compared to the infected group. Our study indicates that the combination of sub-optimal doses of SAG and PV6 may be beneficial for the treatment of SAG resistant visceral leishmaniasis patients.

Designing therapies against experimental visceral leishmaniasis by modulating the membrane fluidity of antigen-presenting cells.

The membrane fluidity of antigen-presenting cells (APCs) has a significant bearing on T-cell-stimulating ability and is dependent on the cholesterol content of the membrane. The relationship, if any, between membrane fluidity and defective cell-mediated immunity in visceral leishmaniasis has been investigated. Systemic administration of cholesterol by liposome delivery (cholesterol liposomes) in Leishmania donovani-infected hamsters was found to cure the infection. Splenic macrophages as a prototype of APCs in infected hamsters had decreased membrane cholesterol and an inability to drive T cells, which was corrected by cholesterol liposome treatment. The effect was cholesterol specific because liposomes made up of the analogue 4-cholesten-3-one provided almost no protection. Infection led to increases in interleukin-10 (IL-10), transforming growth factor beta, and IL-4 signals and concomitant decreases in gamma interferon (IFN-gamma), tumor necrosis factor alpha, and inducible NO synthase signals, which reverted upon cholesterol liposome treatment. The antileishmanial T-cell repertoire, whose expansion appeared to be associated with protection, was presumably type Th1, as shown by enhanced IFN-gamma signals and the predominance of the immunoglobulin G2 isotype. The protected group produced significantly more reactive oxygen species and NO than the infected groups, which culminated in killing of L. donovani parasites. Therefore, cholesterol liposome treatment may be yet another simple strategy to enhance the cell-mediated immune response to L. donovani infection. To our knowledge, this is the first report on the therapeutic effect of cholesterol liposomes in any form of the disease.

Activation of macrophages and lymphocytes by methylglyoxal against tumor cells in the host.

Methylglyoxal is a normal metabolite and has the potential to affect a wide variety of cellular processes. In particular, it can act selectively against malignant cells. The study described herein was to investigate whether methylglyoxal can enhance the non-specific immunity of the host against tumor cells. Methylglyoxal increased the number of macrophages in the peritoneal cavity of both normal and tumor-bearing mice. It also elevated the phagocytic capacity of macrophages in both these groups of animals. This activation of macrophages was brought about by increased production of Reactive Oxygen Intermediates (ROIs) and Reactive Nitrogen Intermediates (RNIs). The possible mechanism for the production of ROIs and RNIs can be attributed to stimulation of the respiratory burst enzyme NADPH oxidase and iNOS, respectively. IFN-gamma, which is a regulatory molecule of iNOS pathway also showed an elevated level by methylglyoxal. TNF-alpha, which is an important cytokine for oxygen independent killing by macrophage also increased by methylglyoxal in both tumor-bearing and non tumor-bearing animals. Methylglyoxal also played a role in the proliferation and cytotoxicity of splenic lymphocytes. In short, it can be concluded that methylglyoxal profoundly stimulates the immune system against tumor cells.

Hybrid cell vaccination resolves Leishmania donovani infection by eliciting a strong CD8+ cytotoxic T-lymphocyte response with concomitant suppression of interleukin-10 (IL-10) but not IL-4 or IL-13.

There is an acute dearth of therapeutic interventions against visceral leishmaniasis that is required to restore an established defective cell-mediated immune response. Hence, formulation of effective immunotherapy requires the use of dominant antigen(s) targeted to elicit a specific antiparasitic cellular immune response. We implemented hybrid cell vaccination therapy in Leishmania donovani-infected BALB/c mice by electrofusing dominant Leishmania antigen kinetoplastid membrane protein 11 (KMP-11)-transfected bone marrow-derived macrophages from BALB/c mice with allogeneic bone marrow-derived dendritic cells from C57BL/6 mice. Hybrid cell vaccine (HCV) cleared the splenic and hepatic parasite burden, eliciting KMP-11-specific major histocompatibility complex class I-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses. Moreover, splenic lymphocytes of HCV-treated mice not only showed the enhancement of gamma interferon but also marked an elevated expression of the Th2 cytokines interleukin-4 (IL-4) and IL-13 at both transcriptional and translational levels. On the other hand, IL-10 production from splenic T cells was markedly suppressed as a result of HCV therapy. CD8+ T-cell depletion completely abrogated HCV-mediated immunity and the anti-KMP-11 CTL response. Interestingly, CD8+ T-cell depletion completely abrogated HCV-induced immunity, resulting in a marked increase of IL-10 but not of IL-4 and IL-13. The present study reports the first implementation of HCV immunotherapy in an infectious disease model, establishing strong antigen-specific CTL generation as a correlate of HCV-mediated antileishmanial immunity that is reversed by in vivo CD8+ T-cell depletion of HCV-treated mice. Our findings might be extended to drug-nonresponsive visceral leishmaniasis patients, as well as against multiple infectious diseases with pathogen-specific immunodominant antigens.