Experimental Visceral Leishmaniasis Research Articles

A molecular signature for IL-10-producing Th1 cells in protozoan parasitic diseases.

Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.

Nanostructured Lipid Carriers as Robust Systems for Lupeol Delivery in the Treatment of Experimental Visceral Leishmaniasis.

Leishmaniasis is a neglected tropical disease that affects millions of people around the world. Available therapy causes severe side effects, has unacceptable prices for some specific formulations, and the existence of drug-resistant parasites limits the use of the currently available arsenal of antiparasitic drugs. Therefore, natural products serve as one of the main sources to develop new and effective alternative drugs against leishmaniasis. In this sense, the present study evaluated the potential of the triterpene Lupeol (Lu) entrapped in nanostructured lipid carriers (NLCs) for the treatment of experimental visceral leishmaniasis. The therapeutic efficacy of Lu or Lu entrapped in NLC (Lu-NLC) was investigated in golden hamsters infected with Leishmania (Leishmania) infantum. Lu-NLC presented a mean particle size of 265.3 ± 4.6 nm, a polydispersity index of <0.25 and a zeta potential of -37.2 ± 0.84 mV; the efficacy of encapsulation was 84.04 ± 0.57%. Studies on hamsters showed that Lu-NLC (5 mg/kg) administered intraperitoneally for 10 consecutive days caused a reduction of 99.9% in the number of parasites in the spleen and liver compared to the untreated infected control. On the contrary, Lu-treated animals (5 mg/kg) had 94.4 and 90.2% less parasites in the spleen and liver, respectively, than the infected group. Additionally, a significant preservation of splenic and hepatic tissues was observed in animals treated with Lu-NLC or Lu. Furthermore, Lu-NLC-treated animals produced high levels of anti-Leishmania IgG2 isotype. These data indicate that NLC potentialized Lu efficacy in experimental visceral leishmaniasis. This work suggests that Lu and nanoformulations carrying this compound may be considered as an important tool to be included in the alternative therapy of leishmaniasis.

Therapeutic potential of rWnt5A in curbing Leishmania donovani infection.

In view of the antagonism of Wnt5A signaling toward microbial pathogens, we were interested in evaluating the therapeutic potential of recombinant Wnt5A (rWnt5A) in curbing Leishmania donovani infection. Initially, using L. donovani-infected RAW 264.7 and peritoneal macrophages, we demonstrated that application of rWnt5A as opposed to the vehicle control to the infected cells significantly dampens L. donovani infection. Inhibition of infection was associated with increase in cell-associated reactive oxygen species (ROS), and blocked by the ROS production inhibitor diphenylene iodonium chloride (DPI). rWnt5A, but not the vehicle control (PBS: phosphate-buffered saline) administration to L. donovani-infected mice appreciably reduced the infection load, and inhibited disease progression as evident from the preservation of splenic white pulp architecture. rWnt5A administration, moreover, led to elevation of both cell-associated ROS and the activation of splenic T cells. Substantial increase in T cell-associated Interleukin-2 (IL-2) and Granzyme B (GRB) upon exposure of splenic lymphocytes harvested from rWnt5A-treated mice to L. donovani-infected RAW 264.7 macrophages in vitro validated the occurrence of L. donovani-responsive T cell activation in vivo. In summary, this study unveils the therapeutic potential of rWnt5A in curbing L. donovani infection and the progression of experimental visceral leishmaniasis possibly through increase in cellular ROS and T cell activation. Accordingly, it opens up a new avenue of investigation into the use of rWnt5A as a therapeutic agent for restraining the progression of drug-resistant L. donovani infection.

Intramuscular Immunization with a Liposomal Multi-Epitope Chimeric Protein Induces Strong Cellular Immune Responses against Visceral Leishmaniasis.

Control of the intracellular parasite Leishmania (L.) requires the activation of strong type 1 cellular immune responses. Towards this goal, in the present study, a multiepitope chimeric protein named LiChimera was encapsulated into cationic liposomes and its protective efficacy against experimental visceral leishmaniasis was investigated. Liposomal LiChimera conferred significant protection against L. infantum as evidenced by the significantly reduced parasite loads in the spleen and liver. Protection detected in Lipo:LiChimera-immunized mice was dependent on the differentiation of long-lasting cellular immune responses and particularly the induction of antigen-specific multifunctional memory CD4+ TH1 and CD8+ T cells that persisted during infection, as evidenced by the persistent high production of IFN-γ and IL-2 and proliferation activity. Notably, protected mice were also characterized by significantly low numbers of non-regulatory CD4+ T cells able to co-produce IFN-γ and IL-10, an important population for disease establishment, as compared to non-immunized control group. Collectively, these results demonstrate that cationic liposomes containing LiChimera can be considered an effective candidate vaccine against visceral leishmaniasis.

Discovery, SAR and mechanistic studies of quinazolinone-based acetamide derivatives in experimental visceral leishmaniasis

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 IC50 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 G1 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.

A novel outlook in the delivery of artemisinin: production and efficacy in experimental visceral leishmaniasis

ABSTRACT The visceral form of leishmaniasis (VL), due to infection by Leishmania infantum, is a neglected tropical disease. The accessible therapeutic options are limited. Artemisinin is an efficient antileishmanial product with poor biological availability that requires high repetition of therapeutic doses in VL. Solid lipid nanoparticles (SLNs) provide targeted delivery, increase bioavailability and reduce toxicity of the traditional therapeutic strategy. The spherical shape artemisinin-loaded SLNs were prepared in a particle diameter of 222.0 ± 14.0 nm. The SLNs showed no particular toxic effect on the parasites, whereas the native artemisinin demonstrated a significant toxicity rate of 31% in viability of the promastigotes at the 250 µg/ml concentration. The therapeutic efficacy of the artemisinin-loaded SLNs was demonstrated in the experimental VL, using the L. infantum-infected BALB/c mice, in the present study. The 10 and 20 mg/kg doses of artemisinin-loaded SLNs showed higher level of antileishmanial efficacy compared with the free artemisinin. There was a significant diminishing of the parasite burden in liver (84.7 ± 4.9%) and spleen (85.0 ± 3.1%) and hepatosplenomegaly by the artemisinin-loaded SLNs treated at 20 mg/kg compared to the free artemisinin. Therefore, the present study supports the superior efficacy of artemisinin-loaded SLNs over the free artemisinin and could be considered as a new therapeutic strategy in the treatment of leishmaniasis.

Leishmania Infection-Induced Proteolytic Processing of SIRPα in Macrophages.

The shedding of cell surface receptors may bring synergistic outcomes through the loss of receptor-mediated cell signaling and competitive binding of the shed soluble receptor to its ligand. Thus, soluble receptors have both biological importance and diagnostic importance as biomarkers in immunological disorders. Signal regulatory protein α (SIRPα), one of the receptors responsible for the 'don't-eat-me' signal, is expressed by myeloid cells where its expression and function are in part regulated by proteolytic cleavage. However, reports on soluble SIRPα as a biomarker are limited. We previously reported that mice with experimental visceral leishmaniasis (VL) manifest anemia and enhanced hemophagocytosis in the spleen accompanied with decreased SIRPα expression. Here, we report increased serum levels of soluble SIRPα in mice infected with Leishmania donovani, a causative agent of VL. Increased soluble SIRPα was also detected in a culture supernatant of macrophages infected with L. donovani in vitro, suggesting the parasite infection promotes ectodomain shedding of SIRPα on macrophages. The release of soluble SIRPα was partially inhibited by an ADAM proteinase inhibitor in both LPS stimulation and L. donovani infection, suggesting a shared mechanism for cleavage of SIRPα in both cases. In addition to the ectodomain shedding of SIRPα, both LPS stimulation and L. donovani infection induced the loss of the cytoplasmic region of SIRPα. Although the effects of these proteolytic processes or changes in SIRPα still remain unclear, these proteolytic regulations on SIRPα during L. donovani infection may explain hemophagocytosis and anemia induced by infection, and serum soluble SIRPα may serve as a biomarker for hemophagocytosis and anemia in VL and the other inflammatory disorders.

Unwinding the mechanism of macrophage repolarization potential of Oceanimonas sp. BPMS22-derived protein protease inhibitor through Toll-like receptor 4 against experimental visceral leishmaniasis.

The Oceanimonas sp. BPMS22-derived protein protease inhibitor (PPI) has been proven to shift macrophages towards an inflammatory state and reduce Leishmania donovani infection in vitro and in vivo. The current study explored and validated the mechanistic aspects of the PPI and Toll-like receptor (TLR) interaction. The PPI exhibited the upregulation of TLR2, TLR4, and TLR6 during treatment which was proven to orchestrate parasite clearance effectively. An in silico study confirmed the high interaction with TLR4 and PPI. Immune blotting confirmed the significant upregulation of TLR4 in macrophages irrespective of L. donovani infection. Pharmacological inhibition and immune blot study confirmed the involvement of the PPI in TLR4-mediated phosphorylation of p38 MAPK and dephosphorylation of ERK1/2, repolarizing to pro-inflammatory macrophage state against experimental visceral leishmaniasis. In addition, in TLR4 knockdown condition, PPI treatment failed to diminish M2 phenotypical markers (CD68, Fizz1, Ym1, CD206, and MSR-2) and anti-inflammatory cytokines (IL-4, IL-10, and TGF-β). Simultaneously, the PPI failed to upregulate the M1 phenotypical markers and pro-inflammatory cytokines (IL-1β, IL-6, IL-12, and IFN-γ) (p < 0.001) during the TLR4 knockdown condition. In the absence of TLR4, the PPI also failed to reduce the parasite load and T-cell proliferation and impaired the delayed-type hypersensitivity response. The absence of pro-inflammatory cytokines was observed during a co-culture study with PPI-treated macrophages (in the TLR4 knockdown condition) with day 10 T-cell obtained from L. donovani-infected mice. This study supports the immunotherapeutic potential of the PPI as it interacted with TLR4 and promoted macrophage repolarization (M2-M1) to restrict the L. donovani parasite burden and helps in the mounting immune response against experimental visceral leishmaniasis.

Thymic alterations resulting from experimental visceral leishmaniasis in a Syrian hamster (Mesocricetus auratus)

BackgroundThe thymus is a lymphoid organ responsible for the development and maturation of T cells, which are part of the Th1, Th2, Th17, and Treg immune responses triggered by visceral leishmaniasis. The maturation and immunological development of T lymphocytes require a bidirectional interaction between the thymic microenvironment of epithelial cells, dendritic cells, and macrophages and the extracellular matrix with differentiating lymphocytes. ObjectivesWe evaluated the morphological characteristics and tissue distribution of hematopoietic and stromal cells in the thymuses of hamsters experimentally infected with Leishmania infantum, aiming to gain an insight into the pathophysiology of the disease. MethodsFifteen hamsters were subjected to intraperitoneal experimental infection with 107L. infantum promastigotes (MHOM/BR/1972/BH46). The animals were divided into three groups, each comprising five infected hamsters, and were then euthanized 15, 60, and 120 days postinfection. The control groups consisted of three groups of five healthy hamsters euthanized simultaneously with the infected ones. Thymic morphology was evaluated through histopathology and the cell composition through immunohistochemistry. We used antibodies to mark mesenchymal cells (anti-vimentin), epithelial cells (anti-cytokeratin), macrophages (anti-MAC387), B lymphocytes (anti-CD79a), and T lymphocytes (anti-CD3). Immunohistochemistry was also used to mark the parasite in the thymus. ResultsInfected and control hamsters showed no difference in thymic morphology and degree of atrophy. After 15 days of infection, CD3 + T lymphocytes in the thymus showed an increase that stabilized over time. At 120 days of infection, we detected a significant decrease in CD79a+ B lymphocytes. The parasite was present in the medullary and corticomedullary regions of 9 out of 15 hamsters. These findings confirm that the presence of a parasite can cause changes in a thymus cell population. However, further studies are needed to evaluate these changes’ effects on the immune response of infected animals.

Combined immunotherapeutic effect of Leishmania-derived recombinant aldolase and Ambisome against experimental visceral leishmaniasis.

Available therapeutics for visceral leishmaniasis (VL), a deadly parasitic infection, are usually associated with inadequate efficacy and adverse aftereffects. Further, the primary site of Leishmania parasite are host macrophages resulting in compromised immunity; ensuing marked T-cell immunosuppression. Such settings emphasize the exploration of chemo-immunotherapeutic strategies for improvising the infected person's immune status with better resolution of infection. Present work employs the immunization of Leishmania-infected hamsters with Leishmania-derived recombinant aldolase (rLdAld) and enolase (rLdEno) proteins in consort with the sub-optimal dose of Ambisome (2.5mg/kg). After the completion of immunization, hamsters were sacrificed on day 60 and 90 post infection and different organ samples were collected to perform immunological assay for evaluating the therapeutic efficacy and modulation in protective cellular immune responses. Combining these proteins, particularly rLdAld with Ambisome (2.5mg/kg), has significantly reduced the parasitic load (∼80%) with remarkable enhancement in DTH and lymphoproliferative responses compared to the infected control and only Ambisome treated groups. Moreover, cytokine levels at RNA and protein levels were noticed to be inclined towards Th-1 phenotype through up-regulation of IFN-γ and TNF-α with significant down-regulation in IL-10 and TGF-β expression, an indication towards the generation of protective immunity against experimental VL. Our experimental findings demonstrated that the chemo-immunotherapeutic approach could be an effective way of controlling human VL infection at minimal dosages of antileishmanial with reduced side-effects and propensity of drug resistance emergence.

Vaccination with Formulation of Nanoparticles Loaded with Leishmania amazonensis Antigens Confers Protection against Experimental Visceral Leishmaniasis in Hamster

Visceral leishmaniasis (VL) is a fatal disease caused by the protozoa Leishmania infantum for which dogs are the main reservoirs. A vaccine against canine visceral leishmaniasis (CVL) could be an important tool in the control of human and CVL by reducing the infection pressure of L. infantum. Despite the CVL vaccine available on the market, the Brazilian Ministry of Health did not implement the use of it in their control programs. In this sense, there is an urgent need to develop more efficient vaccines. In this study, the association between two polymeric nanoformulations, (poly (D, L-lactic) acid (PLA) polymer) loading Leishmania amazonensis antigens, was evaluated as a potential immunobiological agent against VL using golden hamsters as an experimental model. The results indicated that no significant adverse reactions were observed in animals vaccinated with LAPSmP. LAPSmP presented similar levels of total anti-Leishmania IgG as compared to LAPSmG. The LAPSmP and LAPSmG groups showed an intense reduction in liver and spleen parasitic load by qPCR. The LAPSmP and LAPSmG vaccines showed exceptional results, indicating that they may be promising candidates as a VL vaccine.

Design and synthesis of novel halogen rich salicylanilides as potential antileishmanial agents

The available therapeutic treatment for leishmaniasis is inadequate and toxic due to side effects, expensive and emergence of drug resistance. Affordable and safe antileishmanial agents are urgently needed and toward this objective, we synthesized a series of 32 novel halogen rich salicylanilides including niclosamide and oxyclozanide and investigated their antileishmanial activity against amastigotes of Leishmania donovani. In vitro data showed fifteen compounds inhibited intracellular amastigotes with an IC50 of below 5 μM and selectivity index above 10. Among 15 active compounds, 14 and 24 demonstrated better activity with an IC50 of 2.89 μM and 2.09 μM respectively and selectivity index is 18. Compound 24 exhibited significant in vivo antileishmanial efficacy and reduced 65% of the splenic parasite load on day 28th post-treatment in the experimental visceral leishmaniasis golden hamster model. The data suggest that 24 can be a promising lead candidate possessing potential to be developed into a leishmanial drug candidate.

An experimental challenge model for Leishmania donovani in beagle dogs, showing a similar pattern of parasite burden in the peripheral blood and liver.

Leishmania donovani and Leishmania infantum are closely related species. However, the former is considered the causative agent for anthroponotic visceral leishmaniasis (AVL), while the latter is known to be responsible for zoonotic visceral leishmaniasis (ZVL) with dogs as the main reservoir host. Although molecular detection of L. donovani from naturally infected dogs has been reported in AVL endemic areas, the experimental infection of dogs with this species is very limited. Here, we constructed an experimental canine visceral leishmaniasis (CVL) model with L. donovani infection using beagle dogs. During an observation period of 8 months after parasite inoculation, few clinical symptoms were observed in the three inoculated dogs. The overall hematological and biochemical data of the dogs showed normal levels, and there were no remarkable changes in the peripheral CD4+, CD8+, CD25+, or FoxP3+ T cell populations. Liver biopsy sampling was conducted to monitor the parasite burden in the liver. A similar pattern of the amount of mitochondrial kinetoplast DNA was observed in the peripheral blood and liver by real-time PCR analysis. In addition, parasite antigens were detected from the liver biopsy sections by immunohistochemical analysis, further supporting the existence of parasites in the liver. These results showed a subclinical CVL model for L. donovani in beagle dogs with a similar kinetics of parasite burden in the peripheral blood and liver.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00436-022-07681-7.

Leishmania infection-induced multinucleated giant cell formation via upregulation of ATP6V0D2 expression.

Leishmaniasis is caused by infection with protozoan parasites of the genus Leishmania. In both clinical and experimental visceral leishmaniasis, macrophage multinucleation is observed in parasitized tissues. However, the feature and the mechanism of macrophage multinucleation remained unclear. Here, we report that infection of Leishmania donovani, a causative agent of visceral leishmaniasis, induces multinucleation of bone marrow-derived macrophages (BMDMs) in vitro. When these infection-induced multinucleated macrophages were compared with cytokine-induced multinucleated giant cells, the former had higher phagocytic activity on red blood cells but no apparent changes on phagocytosis of latex beads. BMDMs infected with L. donovani had increased expression of ATP6V0D2, one of the components of V-ATPase, which was also upregulated in the spleen of infected mice. Infection-induced ATP6V0D2 localized in a cytoplasmic compartment, which did not overlap with the mitochondria, endoplasmic reticulum, or lysosomes. When ATP6V0D2 expression was recombinantly induced in BMDMs, the formation of multinucleated macrophages was induced as seen in the infected macrophages. Taken together, L. donovani infection induces multinucleation of macrophages via ATP6V0D2 upregulation leading to a unique metamorphosis of the macrophages toward hemophagocytes.

A specific Leishmania infantum polyepitope vaccine triggers Th1-type immune response and protects against experimental visceral leishmaniasis

The development of an immunogenic, effective, and safe vaccine is essential as an alternative for disease control. The present study aimed to evaluate the immunogenicity and efficacy potential of a polyepitope T-cell antigen candidate against visceral leishmaniasis in a murine model. BALB/c mice were immunized with three doses subcutaneously with Poly-T Leish alone or adjuvanted with Saponin plus Monophosphoryl lipid A, with 15-day intervals between doses, and challenged with 107 stationary-phase Leishmania infantum promastigotes via tail vein. Immunogenicity and parasitism in spleen and liver of immunized mice were evaluated 45 days post-challenge. Our results revealed that the immunization with Poly-T Leish and Poly-T Leish/SM increases the percentage of specific T (CD4+ and CD8+) lymphocytes proliferation in vitro after antigen-specific stimulation. Also, Poly-T Leish and Poly-T Leish/SM groups showed a high percentage of IFN-γ and TNF-α-producing T cells, meanwhile, the Poly-T Leish/SM group also showed an increased percentage of multifunctional T cells producing double and triple-positive (IFN-γ+TNF-α+IL-2+) cytokines. The immunization with Poly-T Leish or Poly-T Leish/SM stimulated a decreased IL-4 and IL-10 compared to the Saline and adjuvant group. Poly-T Leish/SM immunized mice exhibit a noteworthy reduction in the parasite burden (spleen and liver) through real-time PCR (96%). Moreover, we observed higher nitrite secretion in 120-hour stimulated-culture supernatant using Griess method. We demonstrated that the Poly-T Leish/SM candidate was potentially immunogenic, providing enhancement of protective immune mechanisms, and conferred protection reducing parasitism. Our candidate was considered potential against visceral leishmaniasis, and eventually, could be tested in phase I and II clinical trials in dogs.

Activation of TLR-pathway to induce host Th1 immune response against visceral leishmaniasis: Involvement of galactosylated-flavonoids

Immunotherapeutic strategies against visceral leishmaniasis (VL) are pertinent because of the emergence of resistance against existing chemotherapy, coupled with their toxicity and high costs. Various bioactive components with potential immunomodulatory activity, such as alkaloids, terpenes, saponins, flavonoids obtained primarily from medicinal plants, have been screened against different disease models. Reports suggested that glycans containing terminal β-galactose can skew host immune response towards Th1 by engaging TLRs. In this study, two synthesized terminal galactose-containing flavones, Quercetin 3-d-galactoside (Q-gal) and Kaempferol 3-O-d-galactoside (K-gal), are profiled in terms of inducing host protective Th1 response in both in vitro &in vivo animal models of experimental VL individually against antimony-resistant & antimony-susceptible Leishmania donovani. Further, we explored that both Q-gal and K-gal induce TLR4 mediated Th1 response to encounter VL. Molecular docking analysis also suggested strong interaction with TLR4 for both the galactosides, with a slightly better binding potential towards Q-gal. Treatment with both Q-gal and K-gal showed significant antileishmanial efficacy. Each considerably diminished the liver and splenic parasite burden 60 days after post-infection (>90% in AG83 infected mice and >87% in GE1F8R infected mice) when administered at a 5 mg/kg/day body-weight dose for ten consecutive days. However, the treatments failed to clear the parasites in the TLR4 deficient C3H/HeJ mice. Treatment with these compounds favors the elevation of TLR4 dependent host protective Th1 cytokines and suppression of disease-promoting IL-10. Q-gal and K-gal also triggered sufficient ROS generation in macrophages to kill intracellular parasites directly.

T Lymphocyte Exhaustion During Human and Experimental Visceral Leishmaniasis.

A key point of immunity against protozoan Leishmania parasites is the development of an optimal T cell response, which includes a low apoptotic rate, high proliferative activity and polyfunctionality. During acute infection, antigen-specific T cells recognize the pathogen resulting in pathogen control but not elimination, promoting the development and the maintenance of a population of circulating effector cells that mount rapid response quickly after re-exposure to the parasite. However, in the case of visceral disease, the functionality of specific T cells is lost during chronic infection, resulting in inferior effector functions, poor response to specific restimulation, and suboptimal homeostatic proliferation, a term referred to as T cell exhaustion. Multiple factors, including parasite load, infection duration and host immunity, affect T lymphocyte exhaustion. These factors contribute to antigen persistence by promoting inhibitory receptor expression and sustained production of soluble mediators, influencing suppressive cell function and the release of endogenous molecules into chronically inflamed tissue. Together, these signals encourage several changes, reprogramming cells into a quiescent state, which reflects disease progression to more severe forms, and development of acquired resistance to conventional drugs to treat the disease. These points are discussed in this review.

Dipeptidylcarboxypeptidase of Leishmania donovani: A potential vaccine molecule against experimental visceral leishmaniasis

Visceral leishmaniasis is one of the deadliest parasitic diseases in the world. In the absence of an efficient and cost-effective drugs, development of an effective vaccine is the need of the day. In spite of several efforts, a successful vaccine against the disease has been elusive. We have evaluated immunoprophylactic efficacy of recombinant dipeptidycarboxypeptidase (rLdDCP), predominantly expressed in amastigotes, in chronic hamster model. rLdDCP induced in vitro lymphoproliferation and NO production in cured hamsters. Immunization with rLdDCP alone, or with BCG, caused significant reduction in parasite load suggesting strong protective response. The molecule also augmented the CMI response as depicted by an increased lymphocyte proliferation, NO production, DTH responses and increased levels of IgG2 in immunized hamsters. The vaccinated hamsters exhibited a surge in IFN-γ, TNF-α, IL-12 and iNOS levels but down-regulation of IL-10 and IL-4. Thus, the results suggest the potentiality of the rLdDCP as a strong candidate vaccine.

Tissue Specific Dual RNA-Seq Defines Host-Parasite Interplay in Murine Visceral Leishmaniasis Caused by Leishmania donovani and Leishmania infantum.

ABSTRACTVisceral leishmaniasis is associated with hepato-splenomegaly and altered immune and hematological parameters in both preclinical animal models and humans. We studied mouse experimental visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani in BALB/c mice using dual RNA-seq to investigate the transcriptional response of host and parasite in liver and spleen. We identified only 4 species-specific parasite expressed genes (SSPEGs; log2FC >1, FDR <0.05) in the infected spleen, and none in the infected liver. For the host transcriptome, we found 789 differentially expressed genes (DEGs; log2FC >1, FDR <0.05) in the spleen that were common to both infections, with IFNγ signaling and complement and coagulation cascade pathways highly enriched, and an additional 286 and 186 DEGs that were selective to L. donovani and L. infantum infection, respectively. Among those, there were network interactions between genes of amino acid metabolism and PPAR signaling in L. donovani infection and increased IL1β and positive regulation of fatty acid transport in L. infantum infection, although no pathway enrichment was observed. In the liver, there were 1,939 DEGs in mice infected with either L. infantum or L. donovani in comparison to uninfected mice, and the most enriched pathways were IFNγ signaling, neutrophil mediated immunity, complement and coagulation, cytokine-chemokine responses, and hemostasis. Additionally, 221 DEGs were selective in L. donovani and 429 DEGs in L. infantum infections. These data show that the host response for these two visceral leishmaniasis infection models is broadly similar, and ∼10% of host DEGs vary in infections with either parasite species.IMPORTANCE Visceral leishmaniasis (VL) is caused by two species of Leishmania parasites, L. donovani in the Old World and L. infantum in the New World and countries bordering the Mediterranean. Although cardinal features such as hepato-splenomegaly and alterations in blood and immune function are evident, clinical presentation may vary by geography, with for example severe bleeding often associated with VL in Brazil. Although animal models of both L. donovani and L. infantum have been widely used to study disease pathogenesis, a direct side-by-side comparison of how these parasites species impact the infected host and/or how they might respond to the stresses of mammalian infection has not been previously reported. Identifying common and distinct pathways to pathogenesis will be important to ensure that new therapeutic or prophylactic approaches will be applicable across all forms of VL.

Persistent Cutaneous Leishmania major Infection Promotes Infection-Adapted Myelopoiesis.

Hematopoietic stem/progenitor cells (HSPC) are responsible for the generation of most immune cells throughout the lifespan of the organism. Inflammation can activate bone marrow HSPCs, leading to enhanced myelopoiesis to replace cells, such as neutrophils, which are attracted to inflamed tissues. We have previously shown that HSPC activation promotes parasite persistence and expansion in experimental visceral leishmaniasis through the increased production of permissive monocytes. However, it is not clear if the presence of the parasite in the bone marrow was required for infection-adapted myelopoiesis. We therefore hypothesized that persistent forms of Leishmania major (cutaneous leishmaniasis) could also activate HSPCs and myeloid precursors in the C57Bl/6 mouse model of intradermal infection in the ear. The accrued influx of myeloid cells to the lesion site corresponded to an increase in myeloid-biased HSPCs in the bone marrow and spleen in mice infected with a persistent strain of L. major, together with an increase in monocytes and monocyte-derived myeloid cells in the spleen. Analysis of the bone marrow cytokine and chemokine environment revealed an attenuated type I and type II interferon response in the mice infected with the persistent strain compared to the self-healing strain, while both strains induced a rapid upregulation of myelopoietic cytokines, such as IL-1β and GM-CSF. These results demonstrate that an active infection in the bone marrow is not necessary for the induction of infection-adapted myelopoiesis, and underline the importance of considering alterations to the bone marrow output when analyzing in vivo host-pathogen interactions.