Comprehensive Proteomics Analysis of the Hemolymph Composition of Sugar-Fed Aedes aegypti Female and Male Mosquitoes.

In arthropods, hemolymph carries immune cells and solubilizes and transports nutrients, hormones, and other molecules that are involved in diverse physiological processes including immunity, metabolism, and reproduction. However, despite such physiological importance, little is known about its composition. We applied mass spectrometry-based label-free quantification approaches to study the proteome of hemolymph perfused from sugar-fed female and male Aedes aegypti mosquitoes. A total of 1403 proteins were identified, out of which 447 of them were predicted to be extracellular. In both sexes, almost half of these extracellular proteins were predicted to be involved in defense/immune response, and their relative abundances (based on their intensity-based absolute quantification, iBAQ) were 37.9 and 33.2%, respectively. Interestingly, among them, 102 serine proteases/serine protease-homologues were identified, with almost half of them containing CLIP regulatory domains. Moreover, proteins belonging to families classically described as chemoreceptors, such as odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), were also highly abundant in the hemolymph of both sexes. Our data provide a comprehensive catalogue of A. aegypti hemolymph basal protein content, revealing numerous unexplored targets for future research on mosquito physiology and disease transmission. It also provides a reference for future studies on the effect of blood meal and infection on hemolymph composition.

Leishmania genetic exchange is mediated by IgM natural antibodies.

Host factors that mediate Leishmania genetic exchange are not well defined. Here we demonstrate that natural IgM (IgMn)1-4 antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. In vivo, we observed a 12-fold increase in hybrid formation in sand flies provided a second blood meal containing IgMn compared with controls. Furthermore, the generation of recombinant progeny from mating hybrids and parental lines were only observed in sand flies provided with IgMn. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.

Optimizing tick artificial membrane feeding for Ixodes scapularis.

Artificial membrane feeding (AMF) is a powerful and versatile technique with a wide range of applications in the study of disease vectors species. Since its first description, AMF has been under constant optimization and standardization for different tick species and life stages. In the USA, Ixodes scapularis is the main vector of tick-borne zoonoses including the pathogens causing Lyme disease in humans and animals. Seeking to improve the overall fitness of I. scapularis adult females fed artificially, here, we have optimized the AMF technique, considerably enhancing attachment rate, engorgement success, egg laying, and egg hatching compared to those described in previous studies. Parameters such as the membrane thickness and the light/dark cycle to which the ticks were exposed were refined to more closely reflect the tick's natural behavior and life cycle. Additionally, ticks were fed on blood only, blood + ATP or blood + ATP + gentamicin. The artificial feeding of ticks on blood only was successful and generated a progeny capable of feeding naturally on a host, i.e., mice. Adding ATP as a feeding stimulant did not improve tick attachment or engorgement. Notably, the administration of gentamicin, an antibiotic commonly used in tick AMF to prevent microbial contamination, negatively impacted Rickettsia buchneri endosymbiont levels in the progeny of artificially fed ticks. In addition, gentamicin-fed ticks showed a reduction in oviposition success compared to ticks artificially fed on blood only, discouraging the use of antibiotics in AMF. Overall, our data suggest that the AMF of adult females on blood only, in association with the natural feeding of their progeny on mice, might be used as an integrated approach in tick rearing, eliminating the use of protected species under the Animal Welfare Act (AWA). Of note, although optimized for I. scapularis adult ticks, I. scapularis nymphs, other tick species, and sand flies could also be fed using the membrane described in this study, indicating that it might be a suitable alternative for the artificial feeding of a variety of hematophagous species.

Sand flies: Basic information on the vectors of leishmaniasis and their interactions with Leishmania parasites.

Blood-sucking arthropods transmit a variety of human pathogens acting as disseminators of the so-called vector-borne diseases. Leishmaniasis is a spectrum of diseases caused by different Leishmania species, transmitted quasi worldwide by sand flies. However, whereas many laboratories focus on the disease(s) and etiological agents, considerably less study the respective vectors. In fact, information on sand flies is neither abundant nor easy to find; aspects including basic biology, ecology, and sand-fly-Leishmania interactions are usually reported separately. Here, we compile elemental information on sand flies, in the context of leishmaniasis. We discuss the biology, distribution, and life cycle, the blood-feeding process, and the Leishmania-sand fly interactions that govern parasite transmission. Additionally, we highlight some outstanding questions that need to be answered for the complete understanding of parasite-vector-host interactions in leishmaniasis.

Some Good and Some Bad: Sand Fly Salivary Proteins in the Control of Leishmaniasis and in Autoimmunity.

Sand flies are hematophagous insects responsible for the transmission of vector-borne diseases to humans. Prominent among these diseases is Leishmaniasis that affects the skin and mucous surfaces and organs such as liver and spleen. Importantly, the function of blood-sucking arthropods goes beyond merely transporting pathogens. The saliva of vectors of disease contains pharmacologically active components that facilitate blood feeding and often pathogen establishment. Transcriptomic and proteomic studies have enumerated the repertoire of sand fly salivary proteins and their potential use for the control of Leishmaniasis, either as biomarkers of vector exposure or as anti-Leishmania vaccines. However, a group of specific sand fly salivary proteins triggers formation of cross-reactive antibodies that bind the ectodomain of human desmoglein 1, a member of the epidermal desmosomal cadherins. These cross-reactive antibodies are associated with skin autoimmune blistering diseases, such as pemphigus, in certain immunogenetically predisposed individuals. In this review, we focus on two different aspects of sand fly salivary proteins in the context of human disease: The good, which refers to salivary proteins functioning as biomarkers of exposure or as anti-Leishmania vaccines, and the bad, which refers to salivary proteins as environmental triggers of autoimmune skin diseases.

Intra- and Extra-Hospital Dissemination of IMP-22-Producing Klebsiella pneumoniae in Northern Portugal: The Breach of the Hospital Frontier Toward the Community.

The emergence of infections (and colonization) with Enterobacteriaceae-producing carbapenemases is a threatening public health problem. In the last decades, we watched an isolated case becoming a brutal outbreak, a sporadic description becoming an endemic problem. The present study aims to highlight the dissemination of IMP-22-producing Klebsiella pneumoniae in the North of Portugal, through the phenotypic and genotypic characterization of isolates collected from hospitalized patients (n=5) and out-patients of the emergency ward of the same acute care hospital (n=2), and isolates responsible for the intestinal colonization of residents in a Long-Term Care Facility (n=4). Pulsed-field gel electrophoresis (PFGE) results, associated with conjugation experiments pointed to a pattern of both vertical and horizontal dissemination. Overall, and complementing other studies that give relevance to IMP-22-producing K. pneumoniae in the clinical settings, here we show for the first time the public health threatening breach of the hospital frontier of this resistance threat, toward the community.

Protective Efficacy in a Hamster Model of a Multivalent Vaccine for Human Visceral Leishmaniasis (MuLeVaClin) Consisting of the KMP11, LEISH-F3+, and LJL143 Antigens in Virosomes, Plus GLA-SE Adjuvant.

Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis, fatal if untreated. Vaccination is the most cost-effective approach to disease control; however, to date, no vaccines against human VL have been made available. This work examines the efficacy of a novel vaccine consisting of the Leishmania membrane protein KMP11, LEISH-F3+ (a recombinant fusion protein, composed of epitopes of the parasite proteins nucleoside hydrolase, sterol-24-c-methyltransferase, and cysteine protease B), and the sand fly salivary protein LJL143, in two dose ratios. The inclusion of the TLR4 agonist GLA-SE as an adjuvant, and the use of virosomes (VS) as a delivery system, are also examined. In a hamster model of VL, the vaccine elicited antigen-specific immune responses prior to infection with Leishmania infantum. Of note, the responses were greater when higher doses of KMP11 and LEISH-F3+ proteins were administered along with the GLA-SE adjuvant and/or when delivered within VS. Remarkably, hamsters immunized with the complete combination (i.e., all antigens in VS + GLA-SE) showed significantly lower parasite burdens in the spleen compared to those in control animals. This protection was underpinned by a more intense, specific humoral response against the KMP11, LEISH-F3+, and LJL143 antigens in vaccinated animals, but a significantly less intense antibody response to the pool of soluble Leishmania antigens (SLA). Overall, these results indicate that this innovative vaccine formulation confers protection against L. infantum infection, supporting the advancement of the vaccine formulation into process development and manufacturing and the conduction of toxicity studies towards future phase I human clinical trials.

Engineering a vector-based pan-Leishmania vaccine for humans: proof of principle.

Leishmaniasis is a spectrum of diseases transmitted by sand fly vectors that deposit Leishmania spp. parasites in the host skin during blood feeding. Currently, available treatment options are limited, associated with high toxicity and emerging resistance. Even though a vaccine for human leishmaniasis is considered an achievable goal, to date we still do not have one available, a consequence (amongst other factors) of a lack of pre-clinical to clinical translatability. Pre-exposure to uninfected sand fly bites or immunization with defined sand fly salivary proteins was shown to negatively impact infection. Still, cross-protection reports are rare and dependent on the phylogenetic proximity of the sand fly species, meaning that the applicability of a sand fly saliva-based vaccine will be limited to a defined geography, one parasite species and one form of leishmaniasis. As a proof of principle of a future vector saliva-based pan-Leishmania vaccine, we engineered through a reverse vaccinology approach that maximizes translation to humans, a fusion protein consisting of immunogenic portions of PdSP15 and LJL143, sand fly salivary proteins demonstrated as potential vaccine candidates against cutaneous and visceral leishmaniasis, respectively. The in silico analysis was validated ex vivo, through T cell proliferation experiments, proving that the fusion protein (administered as a DNA vaccine) maintained the immunogenicity of both PdSP15 and LJL143. Additionally, while no significant effect was detected in the context of L. major transmission by P. duboscqi, this DNA vaccine was defined as partially protective, in the context of L. major transmission by L. longipalpis sand flies. Importantly, a high IFNγ response alone was not enough to confer protection, that mainly correlated with low T cell mediated Leishmania-specific IL-4 and IL-10 responses, and consequently with high pro/anti-inflammatory cytokine ratios. Overall our immunogenicity data suggests that to design a potentially safe vector-based pan-Leishmania vaccine, without geographic restrictions and against all forms of leishmaniasis is an achievable goal. This is why we propose our approach as a proof-of principle, perhaps not only applicable to the anti-Leishmania vector-based vaccines' field, but also to other branches of knowledge that require the design of multi-epitope T cell vaccines with a higher potential for translation.

Heme Oxygenase-1 Induction by Blood-Feeding Arthropods Controls Skin Inflammation and Promotes Disease Tolerance.

Hematophagous vectors lacerate host skin and capillaries to acquire a blood meal, resulting in leakage of red blood cells (RBCs) and inflammation. Here, we show that heme oxygenase-1 (HO-1), a pleiotropic cytoprotective isoenzyme that mitigates heme-mediated tissue damage, is induced after bites of sand flies, mosquitoes, and ticks. Further, we demonstrate that erythrophagocytosis by macrophages, including a skin-residing CD163+CD91+ professional iron-recycling subpopulation, produces HO-1 after bites. Importantly, we establish that global deletion or transient inhibition of HO-1 in mice increases inflammation and pathology following Leishmania-infected sand fly bites without affecting parasite number, whereas CO, an end product of the HO-1 enzymatic reaction, suppresses skin inflammation. This indicates that HO-1 induction by blood-feeding sand flies promotes tolerance to Leishmania infection. Collectively, our data demonstrate that HO-1 induction through erythrophagocytosis is a universal mechanism that regulates skin inflammation following blood feeding by arthropods, thus promoting early-stage disease tolerance to vector-borne pathogens.

Exploring Lutzomyia longipalpis Sand Fly Vector Competence for Leishmania major Parasites.

Lutzomyia longipalpis sand flies are the major natural vector of Leishmania infantum parasites, responsible for transmission of visceral leishmaniasis in the New World. Several experimental studies have demonstrated the ability of Lu. longipalpis to sustain development of different Leishmania species. However, no study had explored in depth the potential vector competence of Lu. longipalpis for Leishmania species other than L. infantum. Here, we show that Lu. longipalpis is a competent vector of L. major parasites, being able to acquire parasites from active cutaneous leishmaniasis lesions, sustain mature infections, and transmit them to naive hosts, causing disease.

Murine infection with bioluminescent Leishmania infantum axenic amastigotes applied to drug discovery.

Leishmaniasis is an important vector-borne neglected tropical disease caused by Leishmania parasites. Current anti-Leishmania chemotherapy is unsatisfactory, justifying the continued search for alternative treatment options. Herein, we demonstrate that luciferase-expressing Leishmania infantum axenic amastigotes, unlike promastigotes, are highly infectious to BALB/c mice and thus generate a robust bioluminescent signal in target organs, such as the liver and the spleen, as early as two weeks after infection. Treatment with the reference drugs amphotericin B and miltefosine was effective at reducing parasite burdens. This model allows the assessment of treatment efficacy using whole-mouse bioluminescence imaging without the need to wait several weeks for spleen infections to be detectable by this non-invasive method. In conclusion, we propose the use of this model in an initial approach to evaluate the treatment efficacy of promising chemical entities without having to sacrifice large numbers of animals or to wait several days for a readout.

Understanding Resistance vs. Susceptibility in Visceral Leishmaniasis Using Mouse Models of Leishmania infantum Infection.

Every year, up to 90,000 new cases of Visceral Leishmaniasis and 30,000 resultant deaths are estimated to occur worldwide. Such numbers give relevance to the continuous study of this complex form of the disease: a zoonosis and an anthroponosis; two known etiological agents (Leishmania infantum and L. donovani, respectively); with an estimated average ratio of 1 symptomatic per 10 asymptomatic individuals; and sometimes associated with atypical clinical presentations. This complexity, which results from a long co-evolutionary process involving vector-host, host-pathogen, and pathogen-vector interactions, is still not completely understood. The determinants of visceralization are not fully defined and the dichotomy resistance vs. susceptibility remains unsolved, translating into obstacles that delay the progress of global disease control. Inbred mouse models, with different susceptibility patterns to Leishmania infection, have been very useful in exploring this dichotomy. BALB/c and C57BL/6 mice were described as susceptible strains to L. donovani visceral infection, while SV/129 was considered resistant. Here, we used these three mouse models, but in the context of L. infantum infection, the other Leishmania species that cause visceral disease in humans, and dynamically compared their local and systemic infection-induced immune responses in order to establish a parallel and to ultimately better understand susceptibility vs. resistance in visceral leishmaniasis. Overall, our results suggest that C57BL/6 mice develop an intermediate "infection-phenotype" in comparison to BALB/c and SV/129 mouse strains, considering both the splenic parasite burden and the determined target organs weights. However, the immune mechanisms associated with the control of infection seem to be different in each mouse strain. We observed that both BALB/c and SV/129, but not C57BL/6 mice, show an infection-induced increase of splenic T follicular helper cells. On the other hand, differences detected in terms of CD21 expression by B cells early after infection, together with the quantified anti-Leishmania specific antibodies, suggest that SV/129 are faster than BALB/c and C57BL/6 mice in the assembly of an efficient B-cell response. Additionally, we observed an infection-induced increase in polyfunctional CD4+ T cells in the resistant SV/129 model, opposing an infection-induced increase in CD4+IL-10+ cells in susceptible BALB/c mice. Our data aligns with the observations reported for L. donovani infection and suggest that not only a single mechanism, but an interaction of several could be necessary for the control of this parasitic disease.

More than just exosomes: distinct Leishmania infantum extracellular products potentiate the establishment of infection.

The use of secretion pathways for effector molecule delivery by microorganisms is a trademark of pathogenesis. Leishmania extracellular vesicles (EVs) were shown to have significant immunomodulatory potential. Still, they will act in conjunction with other released parasite-derived products that might modify the EVs effects. Notwithstanding, the immunomodulatory properties of these non-vesicular components and their influence in the infectious process remains unknown. To address this, we explored both in vitro and in vivo the immunomodulatory potential of promastigotes extracellular material (EXO), obtained as a whole or separated in two different fractions: EVs or vesicle depleted EXO (VDE). Using an air pouch model, we observed that EVs and VDE induced a dose-dependent cell recruitment profile different from the one obtained with parasites, attracting significantly fewer neutrophils and more dendritic cells (DCs). Additionally, when we co-inoculated parasites with extracellular products a drop in cell recruitment was observed. Moreover, in vitro, while VDE (but not EVs) downregulated the expression of DCs and macrophages activation markers, both products were able to diminish the responsiveness of these cells to LPS. Finally, the presence of Leishmania infantum extracellular products in the inoculum promoted a dose-dependent infection potentiation in vivo, highlighting their relevance for the infectious process. In conclusion, our data demonstrate that EVs are not the only relevant players among the parasite exogenous products. This, together with the dose-dependency observed, opens new avenues to the comprehension of Leishmania infectious process. The approach presented here should be exploited to revisit existing data and considered for future studies in other infection models.

Pre-clinical antigenicity studies of an innovative multivalent vaccine for human visceral leishmaniasis.

The notion that previous infection by Leishmania spp. in endemic areas leads to robust anti-Leishmania immunity, supports vaccination as a potentially effective approach to prevent disease development. Nevertheless, to date there is no vaccine available for human leishmaniasis. We optimized and assessed in vivo the safety and immunogenicity of an innovative vaccine candidate against human visceral leishmaniasis (VL), consisting of Virus-Like Particles (VLP) loaded with three different recombinant proteins (LJL143 from Lutzomyia longipalpis saliva as the vector-derived (VD) component, and KMP11 and LeishF3+, as parasite-derived (PD) antigens) and adjuvanted with GLA-SE, a TLR4 agonist. No apparent adverse reactions were observed during the experimental time-frame, which together with the normal hematological parameters detected seems to point to the safety of the formulation. Furthermore, measurements of antigen-specific cellular and humoral responses, generally higher in immunized versus control groups, confirmed the immunogenicity of the vaccine formulation. Interestingly, the immune responses against the VD protein were reproducibly more robust than those elicited against leishmanial antigens, and were apparently not caused by immunodominance of the VD antigen. Remarkably, priming with the VD protein alone and boosting with the complete vaccine candidate contributed towards an increase of the immune responses to the PD antigens, assessed in the form of increased ex vivo CD4+ and CD8+ T cell proliferation against both the PD antigens and total Leishmania antigen (TLA). Overall, our immunogenicity data indicate that this innovative vaccine formulation represents a promising anti-Leishmania vaccine whose efficacy deserves to be tested in the context of the "natural infection".

Health Effects of Overweight and Obesity in 195 Countries over 25 Years.

BACKGROUND: Although the rising pandemic of obesity has received major attention in many countries, the effects of this attention on trends and the disease burden of obesity remain uncertain. METHODS: We analyzed data from 68.5 million persons to assess the trends in the prevalence of overweight and obesity among children and adults between 1980 and 2015. Using the Global Burden of Disease study data and methods, we also quantified the burden of disease related to high body-mass index (BMI), according to age, sex, cause, and BMI in 195 countries between 1990 and 2015. RESULTS: In 2015, a total of 107.7 million children and 603.7 million adults were obese. Since 1980, the prevalence of obesity has doubled in more than 70 countries and has continuously increased in most other countries. Although the prevalence of obesity among children has been lower than that among adults, the rate of increase in childhood obesity in many countries has been greater than the rate of increase in adult obesity. High BMI accounted for 4.0 million deaths globally, nearly 40% of which occurred in persons who were not obese. More than two thirds of deaths related to high BMI were due to cardiovascular disease. The disease burden related to high BMI has increased since 1990; however, the rate of this increase has been attenuated owing to decreases in underlying rates of death from cardiovascular disease. CONCLUSIONS: The rapid increase in the prevalence and disease burden of elevated BMI highlights the need for continued focus on surveillance of BMI and identification, implementation, and evaluation of evidence-based interventions to address this problem. (Funded by the Bill and Melinda Gates Foundation.).

Interleukin-27 Early Impacts Leishmania infantum Infection in Mice and Correlates with Active Visceral Disease in Humans.

The complexity of Leishmania-host interactions, one of the main leishmaniasis issues, is yet to be fully understood. We detected elevated IL-27 plasma levels in European patients with active visceral disease caused by Leishmania infantum, which returned to basal levels after successful treatment, suggesting this cytokine as a probable infection mediator. We further addressed this hypothesis recurring to two classical susceptible visceral leishmaniasis mouse models. BALB/c, but not C57BL/6 mice, showed increased IL-27 systemic levels after infection, which was associated with an upregulation of IL-27p28 expression by dendritic cells and higher parasite burdens. Neutralization of IL-27 in acutely infected BALB/c led to decreased parasite burdens and a transient increase in IFN-γ+ splenic T cells, while administration of IL-27 to C57BL/6 promoted a local anti-inflammatory cytokine response at the site of infection and increased parasite loads. Overall, we show that, as in humans, BALB/c IL-27 systemic levels are infection dependently upregulated and may favor parasite installation by controlling inflammation.

Multidrug-resistant Enterobacteriaceae from indoor air of an urban wastewater treatment plant.

Wastewater treatment plants (WWTPs) have been recognized as sources of bioaerosols that may act as vehicles for dissemination of pathogens and multidrug-resistant (MDR) bacteria. The occurrence of MDR Enterobacteriaceae in indoor air of an urban WWTP was investigated. A possible airborne contamination with extended-spectrum beta-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae was also explored. Fourteen of 39 Enterobacteriaceae isolates were MDR. These isolates were found at all sampling sites, mainly at the secondary sedimentation settings. The highest levels of resistance were detected in three different species: Enterobacter cloacae, Escherichia coli, and Citrobacter freundii. Furthermore, one of the airborne E. coli isolates was phenotypically characterized as an ESBL producer. Additionally, five isolates showed non-susceptibility to at least one carbapenem tested. The presence of genes encoding relevant beta-lactamase types in these ESBL-producing and carbapenem-resistant Enterobacteriaceae isolates was investigated by PCR. Results showed amplification for bla CTX-M and bla OXA. These findings are relevant both in terms of occupational/public health and of environmental dissemination of MDR bacteria.

Disclosing the essentiality of ribose-5-phosphate isomerase B in Trypanosomatids.

Ribose-5-phosphate isomerase (RPI) belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the inter-conversion of D-ribose-5-phosphate and D-ribulose-5-phosphate. Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A, making RPIB worthy of exploration as a potential drug target. Null mutant generation in Leishmania infantum was only possible when an episomal copy of RPIB gene was provided, and the latter was retained both in vitro and in vivo in the absence of drug pressure. This suggests the gene is essential for parasite survival. Importantly, the inability to remove the second allele of RPIB gene in sKO mutants complemented with an episomal copy of RPIB carrying a mutation that abolishes isomerase activity suggests the essentiality is due to its metabolic function. In vitro, sKO promastigotes exhibited no defect in growth, metacyclogenesis or macrophage infection, however, an impairment in intracellular amastigotes' replication was observed. Additionally, mice infected with sKO mutants rescued by RPIB complementation had a reduced parasite burden in the liver. Likewise, Trypanosoma brucei is resistant to complete RPIB gene removal and mice infected with sKO mutants showed prolonged survival upon infection. Taken together our results genetically validate RPIB as a potential drug target in trypanosomatids.

Deception and manipulation: the arms of leishmania, a successful parasite.

Leishmania spp. are intracellular parasitic protozoa responsible for a group of neglected tropical diseases, endemic in 98 countries around the world, called leishmaniasis. These parasites have a complex digenetic life cycle requiring a susceptible vertebrate host and a permissive insect vector, which allow their transmission. The clinical manifestations associated with leishmaniasis depend on complex interactions between the parasite and the host immune system. Consequently, leishmaniasis can be manifested as a self-healing cutaneous affliction or a visceral pathology, being the last one fatal in 85-90% of untreated cases. As a result of a long host-parasite co-evolutionary process, Leishmania spp. developed different immunomodulatory strategies that are essential for the establishment of infection. Only through deception and manipulation of the immune system, Leishmania spp. can complete its life cycle and survive. The understanding of the mechanisms associated with immune evasion and disease progression is essential for the development of novel therapies and vaccine approaches. Here, we revise how the parasite manipulates cell death and immune responses to survive and thrive in the shadow of the immune system.