Leishmania major drives host phagocyte death and cell-to-cell transfer depending on intracellular pathogen proliferation rate.

The virulence of intracellular pathogens relies largely on the ability to survive and replicate within phagocytes but also on release and transfer into new host cells. Such cell-to-cell transfer could represent a target for counteracting microbial pathogenesis. However, our understanding of the underlying cellular and molecular processes remains woefully insufficient. Using intravital 2-photon microscopy of caspase-3 activation in the Leishmania major-infected (L. major-infected) live skin, we showed increased apoptosis in cells infected by the parasite. Also, transfer of the parasite to new host cells occurred directly without a detectable extracellular state and was associated with concomitant uptake of cellular material from the original host cell. These in vivo findings were fully recapitulated in infections of isolated human phagocytes. Furthermore, we observed that high pathogen proliferation increased cell death in infected cells, and long-term residency within an infected host cell was only possible for slowly proliferating parasites. Our results therefore suggest that L. major drives its own dissemination to new phagocytes by inducing host cell death in a proliferation-dependent manner.

Expeditious recruitment of circulating memory CD8 T cells to the liver facilitates control of malaria.

Circulating memory CD8 T cell trafficking and protective capacity during liver-stage malaria infection remains undefined. We find that effector memory CD8 T cells (Tem) infiltrate the liver within 6 hours after malarial or bacterial infections and mediate pathogen clearance. Tem recruitment coincides with rapid transcriptional upregulation of inflammatory genes in Plasmodium-infected livers. Recruitment requires CD8 T cell-intrinsic LFA-1 expression and the presence of liver phagocytes. Rapid Tem liver infiltration is distinct from recruitment to other non-lymphoid tissues in that it occurs both in the absence of liver tissue resident memory "sensing-and-alarm" function and ∼42 hours earlier than in lung infection by influenza virus. These data demonstrate relevance for Tem in protection against malaria and provide generalizable mechanistic insights germane to control of liver infections.

Nanocarrier-enhanced intracellular delivery of benznidazole for treatment of Trypanosoma cruzi infection.

Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), an intracellular pathogen that causes significant morbidity and death among millions in the Americas from Canada to Argentina. Current therapy involves oral administration of the nitroimidazole benznidazole (BNZ), which has serious side effects that often necessitate cessation of treatment. To both avoid off-target side effects and reduce the necessary dosage of BNZ, we packaged the drug within poly(ethylene glycol)-block-poly(propylene sulfide) polymersomes (BNZ-PSs). We show that these vesicular nanocarriers enhanced intracellular delivery to phagocytic cells and tested this formulation in a mouse model of T. cruzi infection. BNZ-PS is not only nontoxic but also significantly more potent than free BNZ, effectively reducing parasitemia, intracellular infection, and tissue parasitosis at a 466-fold lower dose of BNZ. We conclude that BNZ-PS was superior to BNZ for treatment of T. cruzi infection in mice and that further modifications of this nanocarrier formulation could lead to a wide range of custom controlled delivery applications for improved treatment of Chagas disease in humans.

The unicellular eukaryotic parasite Toxoplasma gondii hijacks the migration machinery of mononuclear phagocytes to promote its dissemination.

Toxoplasma gondii is an obligate intracellular protozoan with the ability to infect virtually any type of nucleated cell in warm-blooded vertebrates including humans. Toxoplasma gondii invades immune cells, which the parasite employs as shuttles for dissemination by a Trojan horse mechanism. Recent findings are starting to unveil how this parasite orchestrates the subversion of the migratory functions of parasitised mononuclear phagocytes, especially dendritic cells (DCs) and monocytes. Here, we focus on how T. gondii impacts host cell signalling that regulates leukocyte motility and systemic migration in tissues. Shortly after active parasite invasion, DCs undergo mesenchymal-to-amoeboid transition and adopt a high-speed amoeboid mode of motility. To trigger migratory activation - termed hypermigratory phenotype - T. gondii induces GABAergic signalling, which results in calcium fluxes mediated by voltage-gated calcium channels in parasitised DCs and brain microglia. Additionally, a TIMP-1-CD63-ITGB1-FAK signalling axis and signalling via the receptor tyrosine kinase MET promotes sustained hypermigration of parasitised DCs. Recent reports show that the activated signalling pathways converge on the small GTPase Ras to activate the MAPK Erk signalling cascade, a central regulator of cell motility. To date, three T. gondii-derived putative effector molecules have been linked to hypermigration: Tg14-3-3, TgWIP and ROP17. Here, we discuss their impact on the hypermigratory phenotype of phagocytes. Altogether, the emerging concept suggests that T. gondii induces metastasis-like migratory properties in parasitised mononuclear phagocytes to promote infection-related dissemination.

The host cell secretory pathway mediates the export of Leishmania virulence factors out of the parasitophorous vacuole.

To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV.

Chemical depletion of phagocytic immune cells in Anopheles gambiae reveals dual roles of mosquito hemocytes in anti-Plasmodium immunity.

Mosquito immunity is composed of both cellular and humoral factors that provide protection from invading pathogens. Immune cells known as hemocytes, have been intricately associated with phagocytosis and innate immune signaling. However, the lack of genetic tools has limited hemocyte study despite their importance in mosquito anti-Plasmodium immunity. To address these limitations, we employ the use of a chemical-based treatment to deplete phagocytic immune cells in Anopheles gambiae, demonstrating the role of phagocytes in complement recognition and prophenoloxidase production that limit the ookinete and oocyst stages of malaria parasite development, respectively. Through these experiments, we also define specific subtypes of phagocytic immune cells in An. gambiae, providing insights beyond the morphological characteristics that traditionally define mosquito hemocyte populations. Together, this study represents a significant advancement in our understanding of the roles of mosquito phagocytes in mosquito vector competence and demonstrates the utility of clodronate liposomes as an important tool in the study of invertebrate immunity.

Concanavalin-A displays leishmanicidal activity by inducing ROS production in human peripheral blood mononuclear cells.

The context of the article: Leishmania amazonensis has a wide geographical distribution throughout South American countries and can cause self-healing to severe cases as mucocutaneous or visceral forms. Leishmaniasis presents a balance of inflammatory and anti-inflammatory cytokines which is responsible for promoting the activation of phagocytes, essential to control the infection and lead to tissue repair/resolution of the disease, respectively. Results and discussion: Our model revealed that the treatment with Con-A was capable to stimulate human PBMC cells by increasing the phagocytic capacity and promoting parasite elimination. The pretreatment with Con-A promoted inflammatory (IFN-γ, TNF-α, IL-2 and IL-6) and anti-inflammatory (IL-4 and IL-10) cytokines production, increased the reactive oxygen species (ROS) sinthesys as well as the expression and presence of iNOS enzyme, but not nitric oxide production. Conclusion: Based on the data obtained, it was possible to infer that Con-A induces the ROS production, responsible for eliminating parasites in addition to regulatory cytokines synthesis which are important for disease resolution.

In vitro immunomodulatory effects of microemulsions with levamisole delivery systems on blood phagocytes interacting with Giardia lamblia.

BACKGROUND: Giardiasis is one of the main parasites that infect the gastrointestinal tract of humans, affecting hundreds of millions of people worldwide, particularly in developing countries. Antiparasitics administered to treat giardiasis are inefficient in 20% of the cases, usually because of parasite resistance and side effects. In this scenario, microemulsions are a promising pharmaceutical alternative as carriers of molecules with therapeutic action that stimulate the immune system. METHODS: The study evaluated the effects of a microemulsion delivery system with levamisole hydrochloride on the functional activity of MN phagocytes incubated with G. lamblia. RESULTS: The microemulsion formulated was incorporated with levamisole hydrochloride using distilled water, caprylic/capric triglyceride-Polymol 812®, Sorbitan Oleate-Span 80®, Polysorbate 80 - Tween 80® and 1-butanol. The activity of the microemulsion was analyzed by phagocytosis rate, microbicidal activity, apoptosis rate and intracellular calcium concentration. Phagocytosis rate, microbicidal activity and apoptosis index increased in the microemulsion treatment. The results suggest that the microemulsion improves the therapeutic efficacy of levamisole, increasing the functional activity of phagocytes. CONCLUSIONS: The microemulsion with a levamisole delivery system is therefore an efficient alternative for treating giardiasis, acting as an immunomodulator that probably causes fewer side effects than conventional drugs.

Galleria mellonella hemocytes: A novel phagocytic assay for Leishmania (Viannia) braziliensis.

Galleria mellonella is an excellent invertebrate model for the study of diseases that involve interactions with cells from the innate immune system, since they have an innate immune system capable of recognizing the pathogens. Here we present for the first time, an alternative model for an in vitro phagocytic assay using hemocytes of G. mellonella larvae to study infection by Leishmania (Viannia) braziliensis. We showed that the insect phagocytic cells were able to engulf promastigotes. Furthermore, this infective form differentiated into the amastigote form inside those cells. However, the cells in this model seem resistant to the parasite, since amastigotes were depleted after 24h and NO levels were maintained after infection. Our model opens an avenue of possibilities for new investigations regarding other Leishmania species, mechanisms of invasion and evasion, receptors involved, release of signaling molecules and, above all, it is a novel infection model using invertebrate animals.

A microfluidic cell-trapping device for single-cell tracking of host-microbe interactions.

The impact of cellular individuality on host-microbe interactions is increasingly appreciated but studying the temporal dynamics of single-cell behavior in this context remains technically challenging. Here we present a microfluidic platform, InfectChip, to trap motile infected cells for high-resolution time-lapse microscopy. This approach allows the direct visualization of all stages of infection, from bacterial uptake to death of the bacterium or host cell, over extended periods of time. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae or the intracellular pathogen Mycobacterium marinum. We show that the outcome of such infections is surprisingly heterogeneous, ranging from abortive infection to death of the bacterium or host cell. InfectChip thus provides a simple method to dissect the time-course of host-microbe interactions at the single-cell level, yielding new insights that could not be gleaned from conventional population-based measurements.

The PGE2/IL-10 Axis Determines Susceptibility of B-1 Cell-Derived Phagocytes (B-1CDP) to Leishmania major Infection.

B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. A mononuclear phagocyte derived from B-1 cells (B-1CDP) has been described. As the B-1CDP cells migrate to inflammatory/infectious sites and exhibit phagocytic capacity, the microbicidal ability of these cells was investigated using the Leishmania major infection model in vitro. The data obtained in this study demonstrate that B-1CDP cells are more susceptible to infection than peritoneal macrophages, since B-1CDP cells have a higher number of intracellular amastigotes forms and consequently release a larger number of promastigotes. Exacerbated infection by L. major required lipid bodies/PGE2 and IL-10 by B-1CDP cells. Both infection and the production of IL-10 were decreased when PGE2 production was blocked by NSAIDs. The involvement of IL-10 in this mechanism was confirmed, since B-1CDP cells from IL-10 KO mice are more competent to control L. major infection than cells from wild type mice. These findings further characterize the B-1CDP cells as an important mononuclear phagocyte that plays a previously unrecognized role in host responses to L. major infection, most likely via PGE2-driven production of IL-10.

Stage-specific differential gene expression in Leishmania infantum: from the foregut of Phlebotomus perniciosus to the human phagocyte.

BACKGROUND: Leishmania infantum is the etiological agent of zoonotical visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been recently reported in central Spain. Leishmania spp. parasites are transmitted to the mammalian host by the bite of sand flies. The primary vector of L. infantum in Spain is Phlebotomus perniciosus. For decades, research on these parasites has involved the axenic culture model of the promastigote stage including gene expression profiling studies performed in the post-genome era. Unlike the controversial axenic culturing of amastigotes, promastigote cultures are generally accepted and used, although with the precaution of avoiding excessive culture passage.The primary objective of this differentiation study is to compare the gene expression profiles of promastigotes isolated from the foregut of the sand fly and amastigotes. For this purpose, P. perniciosus sand flies were infected with L. infantum and differentiated promastigotes were extracted by dissection of the foreguts. Shotgun DNA microarray hybridization analyses allowed for transcriptome comparison of these promastigotes with amastigotes obtained by infection of the U937 cell line. The results have been compared with those described in published expression analyses using axenic promastigotes. RESULTS: A total of 277 up-regulated genes were found through this hybridization experiment. The comparison of these particular results with published gene expression profile analyses performed using the same experimental procedure to study cultured promastigotes in stationary phase versus amastigotes revealed considerable differences (approximately 95% of the up-regulated genes were different). We found that the up-regulation rate is lower in amastigotes than in sand fly-derived promastigotes, which is in agreement with the over-expression of genes involved in gene expression regulation and signaling in those promastigote populations. CONCLUSIONS: The up-regulation rate is lower in intracellular amastigotes than in promastigotes obtained from the sand fly gut. This was also reported by us using the promastigote culture model and is an evidence for the hypothesis of promastigote preadaptation towards life in the intracellular environment. Regarding transcript abundance, the set of differentially regulated genes is notably different when using promastigotes from the sand fly foregut instead of axenic cultures.

Immune evasion strategies of pre-erythrocytic malaria parasites.

Malaria is a mosquito-borne infectious disease of humans. It begins with a bite from an infected female Anopheles mosquito and leads to the development of the pre-erythrocytic and blood stages. Blood-stage infection is the exclusive cause of clinical symptoms of malaria. In contrast, the pre-erythrocytic stage is clinically asymptomatic and could be an excellent target for preventive therapies. Although the robust host immune responses limit the development of the liver stage, malaria parasites have also evolved strategies to suppress host defenses at the pre-erythrocytic stage. This paper reviews the immune evasion strategies of malaria parasites at the pre-erythrocytic stage, which could provide us with potential targets to design prophylactic strategies against malaria.

Collective nitric oxide production provides tissue-wide immunity during Leishmania infection.

Nitric oxide (NO) production is critical for the host defense against intracellular pathogens; however, it is unclear whether NO-dependent control of intracellular organisms depends on cell-intrinsic or cell-extrinsic activity of NO. For example, NO production by infected phagocytes may enable these cells to individually control their pathogen burden. Alternatively, the ability of NO to diffuse across cell membranes might be critical for infection control. Here, using a murine ear infection model, we found that, during infection with the intracellular parasite Leishmania major, expression of inducible NO synthase does not confer a cell-intrinsic ability to lower parasite content. We demonstrated that the diffusion of NO promotes equally effective parasite killing in NO-producing and bystander cells. Importantly, the collective production of NO by numerous phagocytes was necessary to reach an effective antimicrobial activity. We propose that, in contrast to a cell-autonomous mode of pathogen control, this cooperative mechanism generates an antimicrobial milieu that provides the basis for pathogen containment at the tissue level.

Mechanisms of cellular invasion by intracellular parasites.

Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.

Evidence of intracellular stages in Trypanosoma (Megatrypanum) theileri in non-phagocytic mammalian cells.

Trypanosoma (subgenus Megatrypanum) theileri was first identified over one hundred years ago, and is a widespread parasite in cattle. Its life cycle within the mammalian host has rarely been reported. Whether there is an intracellular stage in tissues is unknown and such a stage has not been demonstrated experimentally. Intriguingly, using Giemsa staining with light microscopy and transmission electron microscopy examination, we found that the parasite was able not only to attach to cells but also to invade several phagocytic and non-phagocytic mammalian cells. Based on these findings, we conducted further investigations using a special antibody in immunofluorescence confocal images. Moreover, we examined a series of possible events of cell invasion in T. theileri. The results revealed that GM1, a marker of membrane rafts, was implicated in the mechanism of entry by this parasite. After incubation with tissue culture trypomastigotes, the gelatinolytic activity was significantly increased and accumulated at the attachment sites. Using ultrastructural localization detection by CytoTracker live imaging and confocal immunofluorescence microscopy, we found that lysosome fusion and the autophagy pathway were engaged in invaginating processes. T. theileri amastigotes also invaded cells and were enclosed by the lysosomes. Furthermore, tissue-cultured trypomastigotes were found to be capable of triggering intracellular free Ca(2+) transients and TGF-ß-signaling. Our findings that intracellular amastigote stages exist in mammalian cells infected with T. theileri and that the invasion processes involved various host cell components and cell signalings were extremely surprising and warrant further investigation.

Schistosoma mansoni hemozoin modulates alternative activation of macrophages via specific suppression of Retnla expression and secretion.

The trematode Schistosoma mansoni is one of the etiological agents of schistosomiasis, a key neglected tropical disease responsible for an estimated annual loss of 70 million disability-adjusted life years. Hematophagy represents the primary nutrient acquisition pathway of this parasite, but digestion of hemoglobin also liberates toxic heme. Schistosomes detoxify heme via crystallization into hemozoin, which is subsequently regurgitated into the host's circulation. Here we demonstrate that during experimental schistosomiasis, hemozoin accumulating in the mouse liver is taken up by phagocytes at a time coincident with the development of the egg-induced T-helper 2 (Th2) granulomatous immune response. Furthermore, the uptake of hemozoin also coincides with the hepatic expression of markers of alternative macrophage activation. Alternatively activated macrophages are a key effector cell population associated with protection against schistosomiasis, making hemozoin well placed to play an important immunomodulatory role in this disease. To systematically explore this hypothesis, S. mansoni hemozoin was purified and added to in vitro bone marrow-derived macrophage cultures concurrently exposed to cytokines chosen to reflect the shifting state of macrophage activation in vivo. Macrophages undergoing interleukin-4 (IL-4)-induced alternative activation in the presence of hemozoin developed a phenotype specifically lacking in Retnla, a characteristic alternatively activated macrophage product associated with regulation of Th2 inflammatory responses. As such, in addition to its important detoxification role during hematophagy, we propose that schistosome hemozoin also provides a potent immunomodulatory function in the coevolved network of host-parasite relationships during schistosomiasis.

Intracellular pathogen Leishmania donovani activates hypoxia inducible factor-1 by dual mechanism for survival advantage within macrophage.

Recent evidence established a crucial role for mammalian oxygen sensing transcription factor hypoxia inducible factor-1 (HIF-1) in innate immunity against intracellular pathogens. In response to most of these pathogens host phagocytes increase transcription of HIF-1α, the regulatory component of HIF-1 to express various effector molecules against invaders. Leishmania donovani (LD), a protozoan parasite and the causative agent of fatal visceral leishmaniasis resides in macrophages within mammalian host. The mechanism of HIF-1 activation or its role in determining the fate of LD in infected macrophages is still not known. To determine that J774 macrophages were infected with LD and about four-fold increase in HIF-1 activity and HIF-1α expression were detected. A strong increase in HIF-1α expression and nuclear localization was also detected in LD-infected J774 cells, peritoneal macrophages and spleen derived macrophages of LD-infected BALB/c mice. A two-fold increase in HIF-1α mRNA was detected in LD-infected macrophages suggesting involvement of a transcriptional mechanism that was confirmed by promoter activity. We further revealed that LD also induced HIF-1α expression by depleting host cellular iron pool to affect prolyl hydroxylase activity resulting in to stabilization of HIF-1α. To determine the role of HIF-1 on intracellular LD, cells were transfected with HIF-1α siRNA to attenuate its expression and then infected with LD. Although, initial infection rate of LD in HIF-1α attenuated cells was not affected but intracellular growth of LD was significantly inhibited; while, over-expression of stabilized form of HIF-1α promoted intracellular growth of LD in host macrophage. Our results strongly suggest that LD activates HIF-1 by dual mechanism for its survival advantage within macrophage.

Delta-aminolevulinate-induced host-parasite porphyric disparity for selective photolysis of transgenic Leishmania in the phagolysosomes of mononuclear phagocytes: a potential novel platform for vaccine delivery.

Leishmania double transfectants (DTs) expressing the 2nd and 3rd enzymes in the heme biosynthetic pathway were previously reported to show neogenesis of uroporphyrin I (URO) when induced with delta-aminolevulinate (ALA), the product of the 1st enzyme in the pathway. The ensuing accumulation of URO in DT promastigotes rendered them light excitable to produce reactive oxygen species (ROS), resulting in their cytolysis. Evidence is presented showing that the DTs retained wild-type infectivity to their host cells and that the intraphagolysosomal/parasitophorous vacuolar (PV) DTs remained ALA inducible for uroporphyrinogenesis/photolysis. Exposure of DT-infected cells to ALA was noted by fluorescence microscopy to result in host-parasite differential porphyrinogenesis: porphyrin fluorescence emerged first in the host cells and then in the intra-PV amastigotes. DT-infected and control cells differed qualitatively and quantitatively in their porphyrin species, consistent with the expected multi- and monoporphyrinogenic specificities of the host cells and the DTs, respectively. After ALA removal, the neogenic porphyrins were rapidly lost from the host cells but persisted as URO in the intra-PV DTs. These DTs were thus extremely light sensitive and were lysed selectively by illumination under nonstringent conditions in the relatively ROS-resistant phagolysosomes. Photolysis of the intra-PV DTs returned the distribution of major histocompatibility complex (MHC) class II molecules and the global gene expression profiles of host cells to their preinfection patterns and, when transfected with ovalbumin, released this antigen for copresentation with MHC class I molecules. These Leishmania mutants thus have considerable potential as a novel model of a universal vaccine carrier for photodynamic immunotherapy/immunoprophylaxis.