Georgia C. Atella

Lipid droplet formation in leprosy: Toll-like receptor-regulated organelles involved in eicosanoid formation and Mycobacterium leprae pathogenesis

A hallmark of LL is the accumulation of Virchows foamy macrophages. However, the origin and nature of these lipids, as well as their function and contribution to leprosy disease, remain unclear. We herein show that macrophages present in LL dermal lesions are highly positive for ADRP, suggesting that their foamy aspect is at least in part derived from LD (also known as lipid bodies) accumulation induced during ML infection. Indeed, the capacity of ML to induce LD formation was confirmed in vivo via an experimental model of mouse pleurisy and in in vitro studies with human peripheral monocytes and murine peritoneal macrophages. Furthermore, infected cells were shown to propagate LD induction to uninfected, neighboring cells by generating a paracrine signal, for which TLR2 and TLR6 were demonstrated to be essential. However, TLR2 and TLR6 deletions affected LD formation in bacterium‐bearing cells only partially, suggesting the involvement of alternative receptors of the innate immune response besides TLR2/6 for ML recognition by macrophages. Finally, a direct correlation between LD formation and PGE2 production was observed, indicating that ML‐induced LDs constitute intracellular sites for eicosanoid synthesis and that foamy cells may be critical regulators in subverting the immune response in leprosy.

ANTITHROMBOTIC PROPERTIES OF IXOLARIS, A POTENT INHIBITOR OF THE EXTRINSIC PATHWAY OF THE COAGULATION CASCADE

Ixolaris is a two-Kunitz tick salivary gland protein identified in Ixodes scapularis that presents extensive sequence homology to TFPI. It binds to FXa or FX as scaffolds and inhibits tissue factor/FVIIa complex (extrinsic Xnase). Differently from TFPI, ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite, which may also results in decreased FXa activity into the prothrombinase complex. In this report, we show that recombinant (125)I-ixolaris interacts with rat and human FX in plasma and prolongs the prothrombin time (PT) and activated partial thromboplastin time (aPTT) in vitro. We have also investigated the effects of ixolaris in vivo, using a venous thrombosis model. Subcutaneous (s.c.) or intravenous (i.v.) administration of ixolaris in rats caused a dose-dependent reduction in thrombus formation, with complete inhibition attained at 20 microg/kg and 10 microg/kg, respectively. Antithrombotic effects were observed 3 h after s.c. administration of ixolaris and lasted for 24 h thereafter. Ex vivo experiments showed that ixolaris (up to 100 microg/kg) did not affect the aPTT, while the PT was increased by approximately 0.4-fold at the highest ixolaris concentration. Remarkably, effective antithrombotic doses of ixolaris (20 microg/kg) was not associated with bleeding which was significant only at higher doses of the anticoagulant (40 microg/kg). Our experiments demonstrate that ixolaris is an effective and possibly safe antithrombotic agent in vivo.

Leishmanicidal effects of piperine, its derivatives, and analogues on Leishmania amazonensis

Leishmaniasis is a tropical disease caused by protozoan parasites of the genus Leishmania which affects 12 million people worldwide. The discovery of drugs for the treatment of leishmaniasis is a pressing concern in global health programs. The aim of this study aim was to evaluate the leishmanicidal effect of piperine and its derivatives/analogues on Leishmania amazonensis. Our results showed that piperine and phenylamide are active against promastigotes and amastigotes in infected macrophages. Both drugs induced mitochondrial swelling, loose kinetoplast DNA, and led to loss of mitochondrial membrane potential. The promastigote cell cycle was also affected with an increase in the G1 phase cells and a decrease in the S-phase cells, respectively, after piperine and phenylamide treatment. Lipid analysis of promastigotes showed that piperine reduced triglyceride, diacylglycerol, and monoacylglycerol contents, whereas phenylamide only reduced diacylglycerol levels. Both drugs were deemed non toxic to macrophages at 50 μM as assessed by XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt), Trypan blue exclusion, and phagocytosis assays, whereas low toxicity was noted at concentrations higher than 150 μM. None of the drugs induced nitric oxide (NO) production. By contrast, piperine reduced NO production in activated macrophages. The isobologram analysis showed that piperine and phenylamide acted synergistically on the parasites suggesting that they affect different target mechanisms. These results indicate that piperine and its phenylamide analogue are candidates for development of drugs for cutaneous leishmaniasis treatment.

Oogenesis and egg development in triatomines: a biochemical approach

In triatomines, as well as in other insects, accumulation of yolk is a process in which an extra-ovarian tissue, the fat body, produces yolk proteins that are packed in the egg. The main protein, synthesized by the fat body, which is accumulated inside the oocyte, is vitellogenin. This process is also known as vitellogenesis. There are growing evidences in triatomines that besides fat body the ovary also produces yolk proteins. The way these yolk proteins enter the oocyte will be discussed. Yolk is a complex material composed of proteins, lipids, carbohydrates and other minor components which are packed inside the oocyte in an organized manner. Fertilization triggers embryogenesis, a process where an embryo will develop. During embryogenesis the yolk will be used for the construction of a new individual, the first instar nymph. The challenge for the next decade is to understand how and where these egg proteins are used up together with their non-protein components, in pace with the genetic program of the embryo, which enables cell differentiation (early phase of embryogenesis) and embryo differentiation (late phase) inside the egg.

Trypanosoma cruzi Infection Is Enhanced by Vector Saliva through Immunosuppressant Mechanisms Mediated by Lysophosphatidylcholine

ABSTRACT Trypanosoma cruzi, the etiological agent of Chagas disease, is transmitted by bug feces deposited on human skin during a blood meal. However, parasite infection occurs through the wound produced by insect mouthparts. Saliva of the Triatominae bug Rhodnius prolixus is a source of lysophosphatidylcholine (LPC). Here, we tested the role of both triatomine saliva and LPC on parasite transmission. We show that vector saliva is a powerful inducer of cell chemotaxis. A massive number of inflammatory cells were found at the sites where LPC or saliva was inoculated into the skin of mice. LPC is a known chemoattractant for monocytes, but neutrophil recruitment induced by saliva is LPC independent. The preincubation of peritoneal macrophages with saliva or LPC increased fivefold the association of T. cruzi with these cells. Moreover, saliva and LPC block nitric oxide production by T. cruzi-exposed macrophages. The injection of saliva or LPC into mouse skin in the presence of the parasite induces an up-to-sixfold increase in blood parasitemia. Together, our data suggest that saliva of the Triatominae enhances T. cruzi transmission and that some of its biological effects are attributed to LPC. This is a demonstration that a vector-derived lysophospholipid may act as an enhancing factor of Chagas disease.

Extracellular lipid droplets promote hemozoin crystallization in the gut of the blood fluke Schistosoma mansoni

Hemozoin (Hz) is a heme crystal produced upon hemoglobin digestion as the main mechanism of heme disposal in several hematophagous organisms. Here, we show that, in the helminth Schistosoma mansoni, Hz formation occurs in extracellular lipid droplets (LDs). Transmission electron microscopy of adult worms revealed the presence of numerous electron‐lucent round structures similar to LDs in gut lumen, where multicrystalline Hz assemblies were found associated to their surfaces. Female regurgitates promoted Hz formation in vitro in reactions partially inhibited by boiling. Fractionation of regurgitates showed that Hz crystallization activity was essentially concentrated on lower density fractions, which have small amounts of pre‐formed Hz crystals, suggesting that hydrophilic–hydrophobic interfaces, and not Hz itself, play a key catalytic role in Hz formation in S. mansoni. Thus, these data demonstrate that LDs present in the gut lumen of S. mansoni support Hz formation possibly by allowing association of heme to the lipid–water interface of these structures.

Mycobacterium leprae intracellular survival relies on cholesterol accumulation in infected macrophages: a potential target for new drugs for leprosy treatment

We recently showed that Mycobacterium leprae (ML) is able to induce lipid droplet formation in infected macrophages. We herein confirm that cholesterol (Cho) is one of the host lipid molecules that accumulate in ML‐infected macrophages and investigate the effects of ML on cellular Cho metabolism responsible for its accumulation. The expression levels of LDL receptors (LDL‐R, CD36, SRA‐1, SR‐B1, and LRP‐1) and enzymes involved in Cho biosynthesis were investigated by qRT‐PCR and/or Western blot and shown to be higher in lepromatous leprosy (LL) tissues when compared to borderline tuberculoid (BT) lesions. Moreover, higher levels of the active form of the sterol regulatory element‐binding protein (SREBP) transcriptional factors, key regulators of the biosynthesis and uptake of cellular Cho, were found in LL skin biopsies. Functional in vitro assays confirmed the higher capacity of ML‐infected macrophages to synthesize Cho and sequester exogenous LDL‐Cho. Notably, Cho colocalized to ML‐containing phagosomes, and Cho metabolism impairment, through either de novo synthesis inhibition by statins or depletion of exogenous Cho, decreased intracellular bacterial survival. These findings highlight the importance of metabolic integration between the host and bacteria to leprosy pathophysiology, opening new avenues for novel therapeutic strategies to leprosy.

Proliferation and differentiation of Trypanosoma cruzi inside its vector have a new trigger: redox status.

Trypanosoma cruzi proliferate and differentiate inside different compartments of triatomines gut that is the first environment encountered by T. cruzi. Due to its complex life cycle, the parasite is constantly exposed to reactive oxygen species (ROS). We tested the influence of the pro-oxidant molecules H2O2 and the superoxide generator, Paraquat, as well as, metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis. These molecules are heme, hemozoin and urate. We also tested the antioxidants NAC and GSH. Heme induced the proliferation of epimastigotes and impaired the metacyclogenesis. β-hematin, did not affect epimastigote proliferation but decreased parasite differentiation. Conversely, we show that urate, GSH and NAC dramatically impaired epimastigote proliferation and during metacyclogenesis, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. We also quantified the parasite loads in the anterior and posterior midguts and in the rectum of the vector by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. In vivo, the group of vectors fed with reduced molecules showed an increment of trypomastigotes and decreased epimastigotes when analyzed by differential counting. Heme stimulated proliferation by increasing the cell number in the S and G2/M phases, whereas NAC arrested epimastigotes in G1 phase. NAC greatly increased the percentage of trypomastigotes. Taken together, these data show a shift in the triatomine gut microenvironment caused by the redox status may also influence T. cruzi biology inside the vector. In this scenario, oxidants act to turn on epimastigote proliferation while antioxidants seem to switch the cycle towards metacyclogenesis. This is a new insight that defines a key role for redox metabolism in governing the parasitic life cycle.

Lysophosphatidylcholine Triggers TLR2- and TLR4-Mediated Signaling Pathways but Counteracts LPS-Induced NO Synthesis in Peritoneal Macrophages by Inhibiting NF-κB Translocation and MAPK/ERK Phosphorylation

Background Lysophosphatidylcholine (LPC) is the main phospholipid component of oxidized low-density lipoprotein (oxLDL) and is usually noted as a marker of several human diseases, such as atherosclerosis, cancer and diabetes. Some studies suggest that oxLDL modulates Toll-like receptor (TLR) signaling. However, effector molecules that are present in oxLDL particles and can trigger TLR signaling are not yet clear. LPC was previously described as an attenuator of sepsis and as an immune suppressor. In the present study, we have evaluated the role of LPC as a dual modulator of the TLR-mediated signaling pathway. Methodology/Principal Findings HEK 293A cells were transfected with TLR expression constructs and stimulated with LPC molecules with different fatty acid chain lengths and saturation levels. All LPC molecules activated both TLR4 and TLR2-1 signaling, as evaluated by NF-қB activation and IL-8 production. These data were confirmed by Western blot analysis of NF-қB translocation in isolated nuclei of peritoneal murine macrophages. However, LPC counteracted the TLR4 signaling induced by LPS. In this case, NF-қB translocation, nitric oxide (NO) synthesis and the expression of inducible nitric oxide synthase (iNOS) were blocked. Moreover, LPC activated the MAP Kinases p38 and JNK, but not ERK, in murine macrophages. Interestingly, LPC blocked LPS-induced ERK activation in peritoneal macrophages but not in TLR-transfected cells. Conclusions/Significance The above results indicate that LPC is a dual-activity ligand molecule. It is able to trigger a classical proinflammatory phenotype by activating TLR4- and TLR2-1-mediated signaling. However, in the presence of classical TLR ligands, LPC counteracts some of the TLR-mediated intracellular responses, ultimately inducing an anti-inflammatory phenotype; LPC may thus play a role in the regulation of cell immune responses and disease progression.

The major insect lipoprotein is a lipid source to mosquito stages of malaria parasite.

Once mosquito midgut barrier was crossed malaria parasite faces a extensive metabolic developmental program in order to ensure its transmission. In the hemolymph of the mosquito the dynamics of lipid metabolism is conducted by a major lipoprotein, lipophorin (Lp). It was recently shown that Lp is engaged in the mosquito immune response to parasite infection. However, it is not clear if Lp is uptaken by the parasite. Here, we show that oocysts are able to uptake mosquito Lp. The uptake of FITC-labeled Lp was demonstrated in midgut-associated oocysts. Alternatively, to confirm Lp incorporation by oocysts we have conducted another set of experiments with iodinated Lp ((125)I-Lp). Oocysts were able to incorporate (125)I-Lp and the process is both time and temperature dependent. This set of results indicated that no matter oocysts are attached to mosquito midgut wall they bear a lipid sequestering machinery from its surroundings. Phospholipid transfer to sporozoites was also demonstrated. In conclusion, these results demonstrate for the first time that malaria parasite undergoes lipid uptake while in the invertebrate host.