Alan Brito Carneiro

Lutzomyia longipalpis Saliva Triggers Lipid Body Formation and Prostaglandin E2 Production in Murine Macrophages

Background Sand fly saliva contains molecules that modify the hosts hemostasis and immune responses. Nevertheless, the role played by this saliva in the induction of key elements of inflammatory responses, such as lipid bodies (LB, also known as lipid droplets) and eicosanoids, has been poorly investigated. LBs are cytoplasmic organelles involved in arachidonic acid metabolism that form eicosanoids in response to inflammatory stimuli. In this study, we assessed the role of salivary gland sonicate (SGS) from Lutzomyia (L.) longipalpis, a Leishmania infantum chagasi vector, in the induction of LBs and eicosanoid production by macrophages in vitro and ex vivo. Methodology/Principal Findings Different doses of L. longipalpis SGS were injected into peritoneal cavities of C57BL/6 mice. SGS induced increased macrophage and neutrophil recruitment into the peritoneal cavity at different time points. Sand fly saliva enhanced PGE2 and LTB4 production by harvested peritoneal leukocytes after ex vivo stimulation with a calcium ionophore. At three and six hours post-injection, L. longipalpis SGS induced more intense LB staining in macrophages, but not in neutrophils, compared with mice injected with saline. Moreover, macrophages harvested by peritoneal lavage and stimulated with SGS in vitro presented a dose- and time-dependent increase in LB numbers, which was correlated with increased PGE2 production. Furthermore, COX-2 and PGE-synthase co-localized within the LBs induced by L. longipalpis saliva. PGE2 production by macrophages induced by SGS was abrogated by treatment with NS-398, a COX-2 inhibitor. Strikingly, SGS triggered ERK-1/2 and PKC-α phosphorylation, and blockage of the ERK-1/2 and PKC-α pathways inhibited the SGS effect on PGE2 production by macrophages. Conclusion In sum, our results show that L. longipalpis saliva induces lipid body formation and PGE2 production by macrophages ex vivo and in vitro via the ERK-1/2 and PKC-α signaling pathways. This study provides new insights regarding the pharmacological mechanisms whereby L. longipalpis saliva influences the early steps of the hosts inflammatory response.

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.

PPARγ Expression and Function in Mycobacterial Infection: Roles in Lipid Metabolism, Immunity, and Bacterial Killing

Tuberculosis continues to be a global health threat, with drug resistance and HIV coinfection presenting challenges for its control. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a highly adapted pathogen that has evolved different strategies to subvert the immune and metabolic responses of host cells. Although the significance of peroxisome proliferator-activated receptor gamma (PPARγ) activation by mycobacteria is not fully understood, recent findings are beginning to uncover a critical role for PPARγ during mycobacterial infection. Here, we will review the molecular mechanisms that regulate PPARγ expression and function during mycobacterial infection. Current evidence indicates that mycobacterial infection causes a time-dependent increase in PPARγ expression through mechanisms that involve pattern recognition receptor activation. Mycobacterial triggered increased PPARγ expression and activation lead to increased lipid droplet formation and downmodulation of macrophage response, suggesting that PPARγ expression might aid the mycobacteria in circumventing the host response acting as an escape mechanism. Indeed, inhibition of PPARγ enhances mycobacterial killing capacity of macrophages, suggesting a role of PPARγ in favoring the establishment of chronic infection. Collectively, PPARγ is emerging as a regulator of tuberculosis pathogenesis and an attractive target for the development of adjunctive tuberculosis therapies.

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.

Glycoinositolphospholipids from Trypanosomatids Subvert Nitric Oxide Production in Rhodnius prolixus Salivary Glands

Background Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands. Methodology/Principal Findings Here we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities. Conclusions/Significance Taken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.

Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner [1]. To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

Platelet-activating factor (PAF) activates casein kinase 2 in the protozoan parasite Herpetomonas muscarum muscarum

Herpetomonas muscarum muscarum is a flagellate parasite of the family Trypanosomatidae, whose cell differentiation can be triggered by the lipid mediator, PAF. In this study we demonstrate for the first time that PAF effect relies on the activation of casein kinase 2 (CK2). The classical antagonist of PAF receptor, WEB 2086, abrogated PAF-enhanced CK2 activity. CK2 activation by PAF was also inhibited when parasite extracts were assayed in the presence of modulators of PKC, MAPK, and both Ser/Thr and Tyr phosphatases. Finally, a cell permeable inhibitor of CK2 (DRB) suppressed PAF-induced cell differentiation in a dose-dependent manner.

Lysophosphatidylcholine: A Novel Modulator of Trypanosoma cruzi Transmission

Lysophosphatidylcholine is a bioactive lipid that regulates a large number of cellular processes and is especially present during the deposition and infiltration of inflammatory cells and deposition of atheromatous plaque. Such molecule is also present in saliva and feces of the hematophagous organism Rhodnius prolixus, a triatominae bug vector of Chagas disease. We have recently demonstrated that LPC is a modulator of Trypanosoma cruzi transmission. It acts as a powerful chemoattractant for inflammatory cells at the site of the insect bite, which will provide a concentrated population of cells available for parasite infection. Also, LPC increases macrophage intracellular calcium concentrations that ultimately enhance parasite invasion. Finally, LPC inhibits NO production by macrophages stimulated by live T. cruzi, and thus interferes with the immune system of the vertebrate host. In the present paper, we discuss the main signaling mechanisms that are likely used by such molecule and their eventual use as targets to block parasite transmission and the pathogenesis of Chagas disease.

Yolk hydrolases in the eggs of Anticarsia gemmatalis hubner (Lepidoptera: Noctuidae): A role for inorganic polyphosphate towards yolk mobilization

Despite being the main insect pest on soybean crops in the Americas, very few studies have approached the general biology of the lepidopteran Anticarsia gemmatalis and there is a paucity of studies with embryo formation and yolk mobilization in this species. In the present work, we identified an acid phosphatase activity in the eggs of A. gemmatalis (agAP) that we further characterized by means of biochemistry and cell biology experiments. By testing several candidate substrates, this enzyme proved chiefly active with phosphotyrosine; in vitro assays suggested a link between agAP activity and dephosphorylation of egg yolk phosphotyrosine. We also detected strong activity with endogenous and exogenous short chain polyphosphates (PolyP), which are polymers of phosphate residues involved in a number of physiological processes. Both agAP activity and PolyP were shown to initially concentrate in small vesicles clearly distinct from typically larger yolk granules, suggesting subcellular compartmentalization. As PolyP has been implicated in inhibition of yolk proteases, we performed in vitro enzymatic assays with a cysteine protease to test whether it would be inhibited by PolyP. This cysteine protease is prominent in Anticarsia egg homogenates. Accordingly, short chain PolyP was a potent inhibitor of cysteine protease. We thereby suggest that PolyP hydrolysis by agAP is a triggering mechanism of yolk mobilization in A. gemmatalis.

Leishmania infantum lipophosphoglycan induced-Prostaglandin E2production in association with PPAR-γ expression via activation of Toll like receptors-1 and 2

Lipophosphoglycan (LPG) is a key virulence factor expressed on the surfaces of Leishmania promastigotes. Although LPG is known to activate macrophages, the underlying mechanisms resulting in the production of prostaglandin E2 (PGE2) via signaling pathways remain unknown. Here, the inflammatory response arising from stimulation by Leishmania infantum LPG and/or its lipid and glycan motifs was evaluated with regard to PGE2 induction. Intact LPG, but not its glycan and lipid moieties, induced a range of proinflammatory responses, including PGE2 and nitric oxide (NO) release, increased lipid droplet formation, and iNOS and COX2 expression. LPG also induced ERK-1/2 and JNK phosphorylation in macrophages, in addition to the release of PGE2, MCP-1, IL-6, TNF-α and IL-12p70, but not IL-10. Pharmacological inhibition of ERK1/2 and PKC affected PGE2 and cytokine production. Moreover, treatment with rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ), also modulated the release of PGE2 and other proinflammatory mediators. Finally, we determined that LPG-induced PPAR-γ signaling occurred via TLR1/2. Taken together, these results reinforce the role played by L. infantum-derived LPG in the proinflammatory response seen in Leishmania infection.