Circadian Regulation of Leishmania Parasite Internalisation in Macrophages and Downstream Cellular Events.

Leishmania spp. parasites use macrophages as a host cell during infection. As a result, macrophages have a dual role: clearing the parasite as well as acting as host cells. Recently, studies have shown that macrophages harbour circadian clocks, which affect many of their functions such as phagocytosis, receptor expression and cytokine release. Interestingly, Leishmania major infection in hosts was also shown to be under circadian control. Therefore, we decided to investigate what underlies the rhythms of L. major infection within macrophages. Using a culture model of infection of bone marrow-derived macrophages with L. major promastigotes, we show that the parasites are internalised into macrophages with a 24-h variation dependent on a functional circadian clock in the cells. This was associated with a variation in the number of parasites per macrophage. The cell surface expression of parasite receptors was not controlled by the cells' circadian clock. In contrast, the expression of the components of the endocytic pathway, EEA1 and LC3b, varied according to the time of infection. This was paralleled by variations in parasite-induced ROS production as well as cytokine tumour necrosis factor α. In summary, we have uncovered a time-dependent regulation of the internalisation of L. major promastigotes in macrophages, controlled by the circadian clock in these cells, as well as subsequent cellular events in the endocytic pathway, intracellular signalling and cytokine production.

Leishmania major MAPK4 intercepts and redirects CD40 signaling promoting infection.

The parasite Leishmania resides as amastigotes within the macrophage parasitophorous vacuoles inflicting the disease Leishmaniasis. Leishmania selectively modulates mitogen-activated protein kinase (MAPK) phosphorylation subverting CD40-triggered anti-leishmanial functions of macrophages. The mechanism of any pathogen-derived molecule induced host MAPK modulation remains poorly understood. Herein, we show that of the fifteen MAPKs, LmjMAPK4 expression is higher in virulent L. major. LmjMAPK4- detected in parasitophorous vacuoles and cytoplasm- binds MEK-1/2, but not MKK-3/6. Lentivirally-overexpressed LmjMAPK4 augments CD40-activated MEK-1/2-ERK-1/2-MKP-1, but inhibits MKK3/6-p38MAPK-MKP-3, phosphorylation. A rationally-identified LmjMAPK4 inhibitor reinstates CD40-activated host-protective anti-leishmanial functions in L. major-infected susceptible BALB/c mice. These results identify LmjMAPK4 as a MAPK modulator at the host-pathogen interface and establish a pathogen-intercepted host receptor signaling as a scientific rationale for identifying drug targets.

Interleukin-7 potentiates MAPK10-elicited host-protective vaccine against Leishmania donovani.

Leishmania donovani causes the potentially fatal disease visceral leishmaniasis for which neither a vaccine nor an adjuvant for human use exists. Although interleukin-7 (IL-7) is implicated in CD4+ T-cell response stabilization, its anti-leishmanial function is uncertain. Therefore, we examined whether IL-7 would potentiate the efficacy of Leishmania major-expressed MAPK10 (LmjMAPK10; M10)-elicited anti-leishmanial host-protective response. We observed that aligning with IL-7R expression, IL-7 increased IFN-γ-secreting TH1 cell but reduced IL-4-producing TH2 cells and production of IL-10 and TGF-ß effectuating anti-leishmanial functions in susceptible BALB/c mouse-derived macrophages. Co-culturing IL-7-pre-treated L. donovani-infected macrophages with L. donovani-infected BALB/c-derived T cells induced IFN-γ-dominated TH1 type anti-leishmanial function. IL-7 treatment of L. donovani-infected BALB/c mice significantly reduced splenic and hepatic parasite loads. Co-culturing CD4+ T cells from IL to 7-treated mice with L. donovani-infected macrophages reduced amastigote numbers suggesting IL-7-elicited host-protective effector T cells. Priming BALB/c with M10 + IL-7 reduced the splenic parasite burden more effectively than that was observed in M10-primed mice. An enhanced protection against L. donovani infection was accompanied by enhanced IL-12 and IFN-γ, but suppressed IL-10 and IL-4, response and host-protective TH1 and memory T cells. These results indicate IL-7-induced leishmanial antigen-specific memory T cell response that protects a susceptible host against L. donovani infection.

Immunogenic properties of empty pcDNA3 plasmid against zoonotic cutaneous leishmaniasis in mice.

BACKGROUND: Leishmania (L) parasite, the causative agent of zoonotic cutaneous leishmaniasis (ZCL), effectively stimulates the mammalian cells to mount strong humoral responses by enhancing T-helper-2 (Th2)-associated cytokines for its survival. The best strategy to decrease the intensity of infection in the host is induction of cellular immunity. METHODS: We evaluated the effects of the empty bacterial pcDNA3 plasmid on mice infected with L. major and quantified the immune mediators including IFN-γ, IL-4, IL-10, IgG2a, IgG1, arginase activity and nitric oxide (NO) in the mice. Moreover, the footpad lesion size and parasite load were assessed. RESULTS: We observed that pcDNA3 could modulate the immune responses in favor of host cells and decrease the disease severity. Th2- associated mediators, including arginase, IL-4, and IL-10 are downregulated, while cellular responses are upregulated in line with an increase in the levels of nitric oxide (NO) and interfero-gamma (IFN-γ). Interestingly, pcDNA3 induced specific Th1-associated antibodies, IgG2a isotype; however, it suppressed the production of humoral IgG1. The stimulation of the immune response by the empty pcDNA3 is able to shift the immune function to predominant cellular responses caused by Th1, and it had a positive effect on the treatment of zoonotic cutaneous leishmaniasis (ZCL). CONCLUSIONS: Altogether, we introduced the pcDNA3 as a potential interfering factor in the modulation of the immune system against ZCL. Since this vector has been widely used as a control group in different studies, we suggest that the potential function of the empty vector should be deeply assessed, as it exerts anti-parasitic effects on mice infected with L. major.

Resistance Against Leishmania major Infection Depends on Microbiota-Guided Macrophage Activation.

Innate immune cells present a dual role during leishmaniasis: they constitute the first line of host defense but are also the main host cells for the parasite. Response against the infection that results in the control of parasite growth and lesion healing depends on activation of macrophages into a classical activated phenotype. We report an essential role for the microbiota in driving macrophage and monocyte-derived macrophage activation towards a resistance phenotype against Leishmania major infection in mice. Both germ-free and dysbiotic mice showed a higher number of myeloid innate cells in lesions and increased number of infected cells, mainly dermal resident and inflammatory macrophages. Despite developing a Th1 immune response characterized by the same levels of IFN-γ production as the conventional mice, germ-free mice presented reduced numbers of iNOS+ macrophages at the peak of infection. Absence or disturbance of host microbiota impaired the capacity of bone marrow-derived macrophage to be activated for Leishmania killing in vitro, even when stimulated by Th1 cytokines. These cells presented reduced expression of inos mRNA, and diminished production of microbicidal molecules, such as ROS, while presenting a permissive activation status, characterized by increased expression of arginase I and il-10 mRNA and higher arginase activity. Colonization of germ-free mice with complete microbiota from conventional mice rescued their ability to control the infection. This study demonstrates the essential role of host microbiota on innate immune response against L. major infection, driving host macrophages to a resistance phenotype.

The subunits of IL-12, originating from two distinct cells, can functionally synergize to protect against pathogen dissemination in vivo.

Cytokines are typically single gene products, except for the heterodimeric interleukin (IL)-12 family. The two subunits (IL-12p40 and IL-12p35) of the prototype IL-12 are known to be simultaneously co-expressed in activated myeloid cells, which secrete the fully active heterodimer to promote interferon (IFN)γ production in innate and adaptive cells. We find that chimeric mice containing mixtures of cells that can only express either IL-12p40 or IL-12p35, but not both together, generate functional IL-12. This alternate two-cell pathway requires IL-12p40 from hematopoietic cells to extracellularly associate with IL-12p35 from radiation-resistant cells. The two-cell mechanism is sufficient to propel local T cell differentiation in sites distal to the initial infection and helps control systemic dissemination of a pathogen, although not parasite burden, at the site of infection. Broadly, this suggests that early secretion of IL-12p40 monomers by sentinel cells at the infection site may help prepare distal host tissues for potential pathogen arrival.

Network-Based Approaches Reveal Potential Therapeutic Targets for Host-Directed Antileishmanial Therapy Driving Drug Repurposing.

Leishmania parasites are the causal agent of leishmaniasis, an endemic disease in more than 90 countries worldwide. Over the years, traditional approaches focused on the parasite when developing treatments against leishmaniasis. Despite numerous attempts, there is not yet a universal treatment, and those available have allowed for the appearance of resistance. Here, we propose and follow a host-directed approach that aims to overcome the current lack of treatment. Our approach identifies potential therapeutic targets in the host cell and proposes known drug interactions aiming to improve the immune response and to block the host machinery necessary for the survival of the parasite. We started analyzing transcription factor regulatory networks of macrophages infected with Leishmania major. Next, based on the regulatory dynamics of the infection and available gene expression profiles, we selected potential therapeutic target proteins. The function of these proteins was then analyzed following a multilayered network scheme in which we combined information on metabolic pathways with known drugs that have a direct connection with the activity carried out by these proteins. Using our approach, we were able to identify five host protein-coding gene products that are potential therapeutic targets for treating leishmaniasis. Moreover, from the 11 drugs known to interact with the function performed by these proteins, 3 have already been tested against this parasite, verifying in this way our novel methodology. More importantly, the remaining eight drugs previously employed to treat other diseases, remain as promising yet-untested antileishmanial therapies. IMPORTANCE This work opens a new path to fight parasites by targeting host molecular functions by repurposing available and approved drugs. We created a novel approach to identify key proteins involved in any biological process by combining gene regulatory networks and expression profiles. Once proteins have been selected, our approach employs a multilayered network methodology that relates proteins to functions to drugs that alter these functions. By applying our novel approach to macrophages during the Leishmania infection process, we both validated our work and found eight drugs already approved for use in humans that to the best of our knowledge were never employed to treat leishmaniasis, rendering our work as a new tool in the box available to the scientific community fighting parasites.

Glyceraldehyde-3-phosphate dehydrogenase present in extracellular vesicles from Leishmania major suppresses host TNF-alpha expression.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills various physiological roles that are unrelated to its glycolytic function. However, to date, the nonglycolytic function of GAPDH in trypanosomal parasites is absent from the literature. Exosomes secreted from Leishmania, like entire parasites, were found to have a significant impact on macrophage cell signaling and function, indicating cross talk with the host immune system. In this study, we demonstrate that the Leishmania GAPDH (LmGAPDH) protein is highly enriched within the extracellular vesicles (EVs) secreted during infection. To understand the function of LmGAPDH in EVs, we generated control, overexpressed, half-knockout (HKO), and complement cell lines. HKO cells displayed lower virulence compared with control cells when macrophages and BALB/c mice were infected with them, implying a crucial role for LmGAPDH in Leishmania infection and disease progression. Furthermore, upon infection of macrophages with HKO mutant Leishmania and its EVs, despite no differences in TNFA mRNA expression, there was a considerable increase in TNF-α protein expression compared with control, overexpressed, and complement parasites as determined by ELISA, RT-PCR, and immunoblot data. In vitro protein translation studies suggest that LmGAPDH-mediated TNF-α suppression occurs in a concentration-dependent manner. Moreover, mRNA binding assays also verified that LmGAPDH binds to the AU-rich 3'-UTR region of TNFA mRNA, limiting its production. Together, these findings confirmed that the LmGAPDH contained in EVs inhibits TNF-α expression in macrophages during infection via posttranscriptional repression.

Protective CD4+ Th1 cell-mediated immunity is reliant upon execution of effector function prior to the establishment of the pathogen niche.

Intracellular infection with the parasite Leishmania major features a state of concomitant immunity in which CD4+ T helper 1 (Th1) cell-mediated immunity against reinfection coincides with a chronic but sub-clinical primary infection. In this setting, the rapidity of the Th1 response at a secondary site of challenge in the skin represents the best correlate of parasite elimination and has been associated with a reversal in Leishmania-mediated modulation of monocytic host cells. Remarkably, the degree to which Th1 cells are absolutely reliant upon the time at which they interact with infected monocytes to mediate their protective effect has not been defined. In the present work, we report that CXCR3-dependent recruitment of Ly6C+ Th1 effector (Th1EFF) cells is indispensable for concomitant immunity and acute (<4 days post-infection) Th1EFF cell-phagocyte interactions are critical to prevent the establishment of a permissive pathogen niche, as evidenced by altered recruitment, gene expression and functional capacity of innate and adaptive immune cells at the site of secondary challenge. Surprisingly, provision of Th1EFF cells after establishment of the pathogen niche, even when Th1 cells were provided in large quantities, abrogated protection, Th1EFF cell accumulation and IFN-γ production, and iNOS production by inflammatory monocytes. These findings indicate that protective Th1 immunity is critically dependent on activation of permissive phagocytic host cells by preactivated Th1EFF cells at the time of infection.

A sand fly salivary protein acts as a neutrophil chemoattractant.

Apart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

Investigations on the effects of anti-Leishmania major serum on the progression of Leishmania infantum infection in vivo and in vitro - implications of heterologous exposure to Leishmania spp.

Leishmaniasis is a vector-borne parasitic disease caused by protozoa of the genus Leishmania. Twenty different species are known to cause disease in humans with varying degrees of pathology. These diseases are transmitted throughout the geographic range of phlebotomine sandflies, found between the latitudes 50°N and 40°S. This study explores antibody dependent enhancement (ADE) as the cause of disease exacerbation in heterologous exposure of L. major primed mice to L. infantum challenge. BALB/c mice received serum from L. major infected or naive mice. All mice were challenged with L. infantum and tissue parasite burdens were recorded. Animals that received anti-L. major serum exhibited significantly higher parasite burdens. Surprisingly, these parasite burdens were higher than those of mice infected with L. major and challenged with L. infantum. In vitro phagocytosis assays were carried out to measure parasite uptake in the presence of naive vs. anti-L. major serum. J774A.1 murine monocytes were cultured with either L. major or L. infantum in the presence of anti-L. major serum, naive serum, or no serum. Significantly higher rates of L. major uptake by J774A.1 cells occurred in the presence of anti-L. major serum, but no measurable increase of L. infantum phagocytosis was seen. Our results suggest that increased disease severity observed in vivo in mice previously exposed to L. major and challenged with L infantum is not a result of extrinsic ADE. We speculate that intrinsic ADE, due to biased memory T cell responses caused by Fcγ signaling, could account for disease exacerbation seen in the animal model.

MicroRNA155 Plays a Critical Role in the Pathogenesis of Cutaneous Leishmania major Infection by Promoting a Th2 Response and Attenuating Dendritic Cell Activity.

Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4+ Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155-/-) mice. Infection was controlled significantly quicker in the miR155-/- mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155-/- mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ+CD8+ T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major-infected miR155-/- mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major-infected miR155-/- DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155-/- T cells was significantly enhanced in L. major-infected miR155-/- DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function.

Extracellular Vesicles during TriTryps infection: Complexity and future challenges.

The trypanosomatid pathogens Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei, currently grouped as TriTryps, have evolved through the time to overcome the upfront innate immune response and establish the infection in humans adapting many aspects of the parasite-cell host interaction. Extracellular vesicles (EVs) emerge as critical structures carrying different key molecules from parasites and target cells that interact continuously during infection. Current information regarding the structure and composition of these vesicles provide new insights into the primary role of TriTryps-EVs reviewed in this work. Expanding knowledge about these critical vesicular structures will promote advances in basic sciences and in translational applications controlling pathogenesis in the neglected tropical diseases caused by TriTryps.

Evaluation of protection induced by in vitro maturated BMDCs presenting CD8+ T cell stimulating peptides after a heterologous vaccination regimen in BALB/c model against Leishmania major.

Leishmaniasis is a complex vector-borne disease mediated by Leishmania parasite and a strong and long-lasting CD4+ Th1 and CD8+-T cell immunity is required to control the infection. Thus far multivalent subunit vaccines have met this requirement more promisingly. However several full protein sequences cannot be easily arranged in one construct. Instead, new emerging immune-informatics based epitope formulations surpass this restriction. Herein, we aimed to examine the protective potential of a dendritic cell based vaccine presenting epitopes to CD8+ and CD4+-T cells in combination with DNA vaccine encoding the same epitopes against murine cutaneous leishmaniasis. Immature DCs were loaded with epitopes (selected from parasite proteome) in vitro with or without CpG oligonucleotides and were used to immunize BALB/c mice. Peptide coding DNA was used to boost the system and immunological responses were evaluated after Leishmania (L.) major infectious challenge. The pre-challenge response to included epitopes was Th1 polarized which potentially lowered the infection at early time points post-challenge but not at later weeks. Collectively, DC prime-DNA boost was found to be a promising approach for Th1 polarization however the constituent epitopes undoubtedly make a significant contribution in the protection outcome of the vaccine.

Semaphorin 3E Promotes Susceptibility to Leishmania major Infection in Mice by Suppressing CD4+ Th1 Cell Response.

Protective immunity to cutaneous leishmaniasis is mediated by IFN-γ-secreting CD4+ Th1 cells. IFN-γ binds to its receptor on Leishmania-infected macrophages, resulting in their activation, production of NO, and subsequent destruction of parasites. This study investigated the role of Semaphorin 3E (Sema3E) in host immunity to Leishmania major infection in mice. We observed a significant increase in Sema3E expression at the infection site at different timepoints following L. major infection. Sema3E-deficient (Sema3E knockout [KO]) mice were highly resistant to L. major infection, as evidenced by significantly (p < 0.05-0.01) reduced lesion sizes and lower parasite burdens at different times postinfection when compared with their infected wild-type counterpart mice. The enhanced resistance of Sema3E KO mice was associated with significantly (p < 0.05) increased IFN-γ production by CD4+ T cells. CD11c+ cells from Sema3E KO mice displayed increased expression of costimulatory molecules and IL-12p40 production following L. major infection and were more efficient at inducing the differentiation of Leishmania-specific CD4+ T cells to Th1 cells than their wild-type counterpart cells. Furthermore, purified CD4+ T cells from Sema3E KO mice showed increased propensity to differentiate into Th1 cells in vitro, and this was significantly inhibited by the addition of recombinant Sema3E in vitro. These findings collectively show that Sema3E is a negative regulator of protective CD4+ Th1 immunity in mice infected with L. major and suggest that its neutralization may be a potential therapeutic option for treating individuals suffering from cutaneous leishmaniasis.

Use of Immunomagnetic Separation tool in Leishmania promastigotes capture.

Immunomagnetic Separation (IMS) assay has been used for isolation of viable whole organisms. The objective of our work is to produce anti-Leishmania magnetic beads and to assess the efficiency of the IMS technique on Leishmania promastigote capture in culture media. Polyclonal anti-Leishmania antibodies were produced by intravenous injection of viable metacyclic promastigotes of Leishmania (L.) major to rabbit. Purified anti-Leishmania IgG was assessed for their reactivity against both L. major and L. infantum promastigotes then covalently conjugated to magnetic beads and used for IMS. This latter was applied on either L. major promastigote cultures of known concentrations or early stage (24h, 48h, 72h) Novy-MacNeal-Nicolle (NNN) cultures of tissue fluid obtained from cutaneous leishmaniasis (CL) lesions. Promastigotes capture was assessed by either microscopy or qPCR after sample boiling. Indirect immunofluorescence assay showed that polyclonal antibodies reacted against both L. major and L. infantum promastigotes. In 50 µL solution, immunomagnetic beads were able to capture 5 live promastigotes out of 20 and 1050 out of 2500, giving an estimated efficiency of 25-42%. The efficiency of the IMS was lower for a lower number of parasites but still repeatable. On the other hand, IMS-qPCR applied to 14 NNN cultures of confirmed Leishmania lesions showed a higher sensitivity to detect live parasites than routine microscopy observation of promastigotes growth (93% positivity at 72h versus 50% positivity within 2-4 weeks incubation). The estimated number of captured parasites at 72h ranged from 1 to more than 100 parasites / 50 µL liquid phase of culture. These preliminary results open the way for interesting perspectives in the use of cultures for leishmaniasis diagnosis and also for other applications such as Leishmania detection in cultures taken from reservoir animals or sandflies.

The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major.

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.