Phosphatidylserine externalization by apoptotic cells is dispensable for specific recognition leading to innate apoptotic immune responses.

Surface determinants newly expressed by apoptotic cells that are involved in triggering potent immunosuppressive responses, referred to as "innate apoptotic immunity (IAI)" have not been characterized fully. It is widely assumed, often implicitly, that phosphatidylserine, a phospholipid normally cloistered in the inner leaflet of cells and externalized specifically during apoptosis, is involved in triggering IAI, just as it plays an essential role in the phagocytic recognition of apoptotic cells. It is notable, however, that the triggering of IAI in responder cells is not dependent on the engulfment of apoptotic cells by those responders. Contact between the responder and the apoptotic target, on the other hand, is necessary to elicit IAI. Previously, we demonstrated that exposure of protease-sensitive determinants on the apoptotic cell surface are essential for initiating IAI responses; exposed glycolytic enzyme molecules were implicated in particular. Here, we report our analysis of the involvement of externalized phosphatidylserine in triggering IAI. To analyze the role of phosphatidylserine, we employed a panel of target cells that either externalized phosphatidylserine constitutively, independently of apoptosis, or did not, as well as their WT parental cells that externalized the phospholipid in an apoptosis-dependent manner. We found that the externalization of phosphatidylserine, which can be fully uncoupled from apoptosis, is neither sufficient nor necessary to trigger the profound immunomodulatory effects of IAI. These results reinforce the view that apoptotic immunomodulation and phagocytosis are dissociable and further underscore the significance of protein determinants localized to the cell surface during apoptosis in triggering innate apoptotic immunity.

Human Endogenous Retrovirus, SARS-CoV-2, and HIV Promote PAH via Inflammation and Growth Stimulation.

Pulmonary arterial hypertension (PAH) is a pulmonary vascular disease characterized by the progressive elevation of pulmonary arterial pressures. It is becoming increasingly apparent that inflammation contributes to the pathogenesis and progression of PAH. Several viruses are known to cause PAH, such as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), human endogenous retrovirus K(HERV-K), and human immunodeficiency virus (HIV), in part due to acute and chronic inflammation. In this review, we discuss the connections between HERV-K, HIV, SARS-CoV-2, and PAH, to stimulate research regarding new therapeutic options and provide new targets for the treatment of the disease.

Signal Transduction during Metabolic and Inflammatory Reprogramming in Pulmonary Vascular Remodeling.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by (mal)adaptive remodeling of the pulmonary vasculature, which is associated with inflammation, fibrosis, thrombosis, and neovascularization. Vascular remodeling in PAH is associated with cellular metabolic and inflammatory reprogramming that induce profound endothelial and smooth muscle cell phenotypic changes. Multiple signaling pathways and regulatory loops act on metabolic and inflammatory mediators which influence cellular behavior and trigger pulmonary vascular remodeling in vivo. This review discusses the role of bioenergetic and inflammatory impairments in PAH development.

Inhibitory effects promoted by 5'-nucleotides on the ecto-3'-nucleotidase activity of Leishmania amazonensis.

The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3'-nucleotidase/nuclease (3'NT/NU), which is able to hydrolyze extracellular 3'-monophosphorylated nucleosides and nucleic acids. 3'NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3'NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3'-nucleotidase (3'NT) activity of L. amazonensis by several nucleotides. It was observed that 3'NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5'-GMP on the 3'NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5'-GMP is able to reverse the stimulation promoted by 3'-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3'NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target.

Effects of platelet-activating factor on the interaction of Trypanosoma cruzi with Rhodnius prolixus.

We investigated the effects of platelet-activating factor (PAF) on the interaction of Trypanosoma cruzi with Rhodnius prolixus. The parasites (epimastigotes) were treated with PAF and/or WEB 2086 (PAF antagonist) for 1 h prior to the interaction experiments. PAF stimulated both in vivo and ex vivo interactions between T. cruzi and R. prolixus while WEB 2086 abrogated these effects. PAF-treated epimastigotes also showed an increase in surface negativity and in the amount of surface sialic acid. Neither of these effects was observed when the epimastigotes were treated with neuraminidase following PAF treatment. In the ex vivo interaction experiments, the number of epimastigotes bound to the midguts of the insects was reduced when the epimastigotes had been treated with neuraminidase. We conclude that PAF modulates the interaction of T. cruzi with R. prolixus by altering the amount of sialyl residues at the surface of the parasite.

Sphingosine Kinase 1 Regulates the Pulmonary Vascular Immune Response.

The aberrant proliferation of pulmonary artery smooth muscle (PASMCs) cells is a defining characteristic of pulmonary arterial hypertension (PAH) and leads to increased vascular resistance, elevated pulmonary pressure, and right heart failure. The sphingosine kinase 1 (SPHK1)/sphingosine-1 phosphate/sphingosine-1 phosphate receptor 2 pathway promotes vascular remodeling and induces PAH. The aim of this study was to identify genes and cellular processes that are modulated by over-expression of SPHK1 in human PASMCs (hPASMCs). RNA was purified and submitted for RNA sequencing to identify differentially expressed genes. Using a corrected p-value threshold of <0.05, there were 294 genes significantly up-regulated while 179 were significantly down-regulated. Predicted effects of these differentially expressed genes were evaluated using the freeware tool Enrichr to assess general gene set over-representation (enrichment) and ingenuity pathway analysis (IPA™) for upstream regulator predictions. We found a strong change in genes that regulated the cellular immune response. IL6, STAT1, and PARP9 were elevated in response to SPHK1 over-expression in hPASMCs. The gene set enrichment mapped to a few immune-modulatory signaling networks, including IFNG. Furthermore, PARP9 and STAT1 protein were elevated in primary hPASMCs isolated from PAH patients. In conclusion, these data suggest a role of Sphk1 regulates pulmonary vascular immune response in PAH.

Platelet-activating factor-like activity isolated from Trypanosoma cruzi.

Platelet-activating factor is a phospholipid mediator that exhibits a wide variety of physiological and pathophysiological effects, including induction of inflammatory response, chemotaxis and cellular differentiation. Trypanosoma cruzi, the etiological agent of Chagas' disease, is transmitted by triatomine insects and while in the triatomine midgut the parasite differentiates from a non-infective epimastigote stage into the pathogenic trypomastigote metacyclic form. We have previously demonstrated that platelet activating factor triggers in vitro cell differentiation of T. cruzi. Here we show a platelet activating factor-like activity isolated from lipid extract of T. cruzi epimastigotes incubated in the presence of [14C]acetate. Trypanosoma cruzi-platelet activating factor-like lipid induced the aggregation of rabbit platelets, which was prevented by platelet activating factor-acetylhydrolase. Mouse macrophage infection by T. cruzi was stimulated when epimastigotes were kept for 5 days in the presence of T. cruzi-platelet activating factor, before interacting with the macrophages. The differentiation of epimastigotes into metacyclic trypomastigotes was also triggered by T. cruzi-platelet activating factor. These effects were abrogated by a platelet activating factor antagonist, WEB 2086. Polyclonal antibody raised against mouse platelet activating factor receptor showed labelling for T. cruzi epimastigotes using immunoblotting and immunofluorescence assays. These data suggest that T. cruzi contain the components of an autocrine platelet activating factor-like ligand-receptor system that modulates cell differentiation towards the infectious stage.

Structural and functional analysis of a platelet-activating lysophosphatidylcholine of Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF. CONCLUSIONS/SIGNIFICANCE: Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions.

Possible roles of ectophosphatases in host-parasite interactions.

The interaction and survival of pathogens in hostile environments and in confrontation with host immune responses are important mechanisms for the establishment of infection. Ectophosphatases are enzymes localized at the plasma membrane of cells, and their active sites face the external medium rather than the cytoplasm. Once activated, these enzymes are able to hydrolyze phosphorylated substrates in the extracellular milieu. Several studies demonstrated the presence of surface-located ecto-phosphatases in a vast number of pathogenic organisms, including bacteria, protozoa, and fungi. Little is known about the role of ecto-phosphatases in host-pathogen interactions. The present paper provides an overview of recent findings related to the virulence induced by these surface molecules in protozoa and fungi.

Biochemical properties of Candida parapsilosis ecto-5'-nucleotidase and the possible role of adenosine in macrophage interaction.

Candida parapsilosis is considered to be an emerging fungal pathogen because it is associated with an increasing range of infections. In this work, we biochemically characterized ecto-5'-nucleotidase activity on the surface of living, intact C. parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 5'-AMP at a rate of 52.44 ± 7.01 nmol Pi h(-1) 10(-7) cells. 5'-AMP, 5'-IMP and 5'-UMP were hydrolyzed at similar rates, whereas 5'-GMP and 5'-CMP hydrolyzed at lower rates. Enzyme activity was increased by about 42% with addition of Mg(2+) or Ca(2+), and the optimum pH was in the acidic range. An inhibitor of phosphatase activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however, as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited the activity in a dose-dependent manner. The results indicated that the existence of an ecto-5'-nucleotidase could play a role in the control of extracellular nucleotide concentrations.

Leishmania amazonensis: Biological and biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities.

The presence of Leishmania amazonensis ecto-nucleoside triphosphate triphosphohydrolase activities was demonstrated using antibodies against different NTPDase members by Western blotting, flow cytometry, and immunoelectron microscopy analysis. Living promastigote cells sequentially hydrolyzed the ATP molecule generating ADP, AMP, and adenosine, indicating that this surface enzyme may play a role in the salvage of purines from the extracellular medium. The L. amazonensis ecto-NTPDase activities were insensitive to Triton X-100, but they were enhanced by divalent cations, such as Mg(2+). In addition, the ecto-NTPDase activities decreased with time for 96 h when promastigotes were grown in vitro. On the other hand, these activities increased considerably when measured in living amastigote forms. Furthermore, the treatment with adenosine, a mediator of several relevant biological phenomena, induced a decrease in the reactivity with anti-CD39 antibody, raised against mammalian E-NTPDase, probably because of down regulation in the L. amazonensis ecto-NTPDase expression. Also, adenosine and anti-NTPDase antibodies induced a significant diminishing in the interaction between promastigotes of L. amazonensis and mouse peritoneal macrophages.