Polyunsaturated fatty acids alter the formation of lipid droplets and eicosanoid production in Leishmania promastigotes

BACKGROUND The knowledge about eicosanoid metabolism and lipid droplet (LD) formation in the Leishmania is very limited and new approaches are needed to identify which bioactive molecules are produced of them. OBJECTIVES Herein, we compared LDs and eicosanoids biogenesis in distinct Leishmania species which are etiologic agents of different clinical forms of leishmaniasis. METHODS For this, promastigotes of Leishmania amazonensis, L. braziliensis and L. infantum were stimulated with polyunsaturated fatty acids (PUFA) and LD and eicosanoid production was evaluated. We also compared mutations in structural models of human-like cyclooxygenase-2 (GP63) and prostaglandin F synthase (PGFS) proteins, as well as the levels of these enzymes in parasite cell extracts. FINDINGS PUFAs modulate the LD formation in L. braziliensis and L. infantum. Leishmania spp with equivalent tissue tropism had same protein mutations in GP63 and PGFS. No differences in GP63 production were observed among Leishmania spp, however PGFS production increased during the parasite differentiation. Stimulation with arachidonic acid resulted in elevated production of hydroxyeicosatetraenoic acids compared to prostaglandins. MAIN CONCLUSIONS Our data suggest LD formation and eicosanoid production are distinctly modulated by PUFAS dependent of Leishmania species. In addition, eicosanoid-enzyme mutations are more similar between Leishmania species with same host tropism.

Coagulopathy and the humoral response against viral proteins in patients at different stages of COVID-19

BACKGROUND Patients with severe coronavirus disease 2019 (COVID-19) often present with coagulopathies and have high titres of circulating antibodies against viral proteins. OBJECTIVES Herein, we evaluated the association between D-dimer and circulating immunoglobulin levels against viral proteins in patients at different clinical stages of COVID-19. METHODS For this, we performed a cross-sectional study involving patients of the first wave of COVID-19 clinically classified as oligosymptomatic (n = 22), severe (n = 30), cured (n = 27) and non-infected (n = 9). Next, we measured in the plasma samples the total and fraction of immunoglobulins against the nucleoprotein (NP) and the receptor-binding domain (RBD) of the spike proteins by enzyme-linked immunosorbent assay (ELISA) assays. FINDINGS Patients with severe disease had a coagulation disorder with high levels of D-dimer as well as circulating IgG against the NP but not the RBD compared to other groups of patients. In addition, high levels of D-dimer and IgG against the NP and RBD were associated with disease severity among the patients in this study. MAIN CONCLUSIONS Our data suggest that IgG against NP and RBD participates in the worsening of COVID-19. Although the humoral response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is partially understood, and more efforts are needed to clarify gaps in the knowledge of this process.

Associations between TGF-ß1 Levels and Markers of Hemolysis, Inflammation, and Tissue Remodeling in Pediatric Sickle Cell Patients.

Transforming growth factor beta (TGF-ß) is a cytokine with important involvement in biological processes related to the pathogenesis of sickle cell disease (SCD), including endothelial and vascular dysfunction, inflammation, and hematopoietic homeostasis. This study is aimed at investigating associations between levels of TGF-ß1 and classical laboratory biomarkers and inflammatory mediators, as well as the tissue inhibitor of metalloproteases-1 (TIMP-1) and matrix metalloproteinase-9 (MMP-9), in pediatric patients (n = 123) with SCD in steady state: 84 with sickle cell anemia (HbSS) and 39 with hemoglobin SC disease (HbSC). A healthy control (HC) group of 59 individuals was also included. Hematological and biochemical analyses were carried out using electronic methods. TGF-ß1, TIMP-1, and MMP-9 plasma quantifications were performed by ELISA. TGF-ß1 plasma levels were higher in HbSS individuals than in HbSC and HC. In individuals with HbSS, TGF-ß1 levels were positively correlated with red blood cells, hemoglobin, hematocrit, platelets, and TIMP-1. In addition, HbSS individuals with TGF-ß1 levels above the median (≥72.29 ng/mL) also presented increased monocyte counts and decreased albumin levels. In patients with HbSC, TGF-ß1 levels were positively correlated with leukocytes, eosinophils, lymphocytes, monocytes, and platelets, as well as levels of TIMP-1, VLDL-C, triglycerides, heme, and AST. Additionally, HbSC individuals with TGF-ß1 levels above the median (≥47.80 ng/mL) presented increased leukocyte and platelet counts, as well as increased levels of triglycerides, VLDL-C, MMP-9, and TIMP-1, and decreased HDL-C. Our findings suggest that TGF-ß1 may play important roles in vascular remodeling, vasculopathy, angiogenesis, and inflammation in pediatric patients with SCD.

Lipid droplets of protozoan parasites: survival and pathogenicity

Lipid droplets (LDs; lipid bodies) are intracellular sites of lipid storage and metabolism present in all cell types. Eukaryotic LDs are involved in eicosanoid production during several inflammatory conditions, including infection by protozoan parasites. In parasites, LDs play a role in the acquisition of cholesterol and other neutral lipids from the host. The number of LDs increases during parasite differentiation, and the biogenesis of these organelles use specific signaling pathways involving protein kinases. In addition, LDs are important in cellular protection against lipotoxicity. Recently, these organelles have been implicated in eicosanoid and specialised lipid metabolism. In this article, we revise the main functions of protozoan parasite LDs and discuss future directions in the comprehension of these organelles in the context of pathogen virulence.

Leishmania infantum lipophosphoglycan induced-Prostaglandin E2 production 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.

Degranulating Neutrophils Promote Leukotriene B4 Production by Infected Macrophages To Kill Leishmania amazonensis Parasites.

Neutrophils mediate early responses against pathogens, and they become activated during endothelial transmigration toward the inflammatory site. In the current study, human neutrophils were activated in vitro with immobilized extracellular matrix proteins, such as fibronectin (FN), collagen, and laminin. Neutrophil activation by FN, but not other extracellular matrix proteins, induces the release of the granules' contents, measured as matrix metalloproteinase 9 and neutrophil elastase activity in culture supernatant, as well as reactive oxygen species production. Upon contact with Leishmania amazonensis-infected macrophages, these FN-activated neutrophils reduce the parasite burden through a mechanism independent of cell contact. The release of granule proteases, such as myeloperoxidase, neutrophil elastase, and matrix metalloproteinase 9, activates macrophages through TLRs, leading to the production of inflammatory mediators, TNF-α and leukotriene B4 (LTB4), which are involved in parasite killing by infected macrophages. The pharmacological inhibition of degranulation reverted this effect, abolishing LTB4 and TNF production. Together, these results suggest that FN-driven degranulation of neutrophils induces the production of LTB4 and TNF by infected macrophages, leading to the control of Leishmania infection.

Prostaglandin E2/leukotriene B4 balance induced by Lutzomyia longipalpis saliva favors Leishmania infantum infection.

BACKGROUND: Eicosanoids and sand fly saliva have a critical role in the Leishmania infection. Here, we evaluated the effect of Lutzomyia longipalpis salivary gland sonicate (SGS) on neutrophil and monocyte recruitment and activation of eicosanoid production in a murine model of inflammation. METHODS: C57BL/6 mice were inoculated intraperitonealy with Lutzomyia longipalpis SGS or Leishmania infantum or both, followed by analyses of cell recruitment, parasite load and eicosanoid production. RESULTS: Intraperitoneal injection of Lutzomyia longipalpis SGS together with Leishmania infantum induced an early increased parasite viability in monocytes and neutrophils. L. longipalpis SGS increased prostaglandin E2 (PGE2), but reduced leukotriene B4 (LTB4) production ex vivo in peritoneal leukocytes. In addition, the pharmacological inhibition of cyclooxygenase 2 (COX-2) with NS-398 decreased parasite viability inside macrophages during Leishmania infection in the presence of L. longipalpis SGS arguing that PGE2 production is associated with diminished parasite killing. CONCLUSIONS: These findings indicate that L. longipalpis SGS is a critical factor driving immune evasion of Leishmania through modulation of PGE2/LTB4 axis, which may represent an important mechanism on establishment of the infection.

Role of prostaglandin F2α production in lipid bodies from Leishmania infantum chagasi: insights on virulence.

Lipid bodies (LB; lipid droplets) are cytoplasmic organelles involved in lipid metabolism. Mammalian LBs display an important role in host-pathogen interactions, but the role of parasite LBs in biosynthesis of prostaglandin F2α (PGF2α) has not been investigated. We report herein that LBs increased in abundance during development of Leishmania infantum chagasi to a virulent metacyclic stage, as did the expression of PGF2α synthase (PGFS). The amount of parasite LBs and PGF2α were modulated by exogenous arachidonic acid. During macrophage infection, LBs were restricted to parasites inside the parasitophorous vacuoles (PV). We detected PGF2α receptor (FP) on the Leishmania PV surface. The blockage of FP with AL8810, a selective antagonist, hampered Leishmania infection, whereas the irreversible inhibition of cyclooxygenase with aspirin increased the parasite burden. These data demonstrate novel functions for parasite-derived LBs and PGF2α in the cellular metabolism of Leishmania and its evasion of the host immune response.

Corpúsculos lipídicos e eicosanoides nos momentos iniciais da infecção com leishmania infantum chagasi / Eicosanoids and lipid bodies in the early stages of infection with Leishmania infantum chagasi

Corpúsculos lipídicos são organelas citoplasmáticas envolvidas na produção de eicosanoides em leucócitos. Eicosanoides como as prostaglandinas têm sido envolvidos no controle da resposta inflamatória e imunológica. A saliva de Lutzomyia longipalpis participa do estabelecimento e desenvolvimento da doença pela modulação das respostas hemostática, imunológica e inflamatória do hospedeiro favorecendo a infecção. Entretanto, o papel dos eicosanoides nos momentos iniciais da infecção por Leishmania ainda não foi esclarecido, assim como a participação da saliva neste contexto. Aqui, nós investigamos o papel dos eicosanoides induzidos pela saliva de L. longipalpis e produzidos pela Leishmania infantum chagasi na infecção. O sonicado de glândula salivar (SGS) de L. longipalis induziu um aumento no número de CLs em macrófagos de maneira dose e tempo dependente, o qual esteve correlacionado com o aumento de PGE2 nos sobrenadante de cultura. As enzimas COX-2 e PGE- intase foram co-localizadas nos CLs induzidos pela saliva e a produção de PGE2 foi reduzida pelo tratamento com NS-398, um inibidor de COX-2. Nós verificamos que o SGS rapidamente estimulou a fosforilação de ERK-1/2 e PKC-α e a inibição farmacológica dessas vias inibiu a produção de PGE2 pelos macrófagos estimulados com SGS. Em seguida, nós avaliamos o efeito da saliva de L. longipalpis sobre a produção de eicosanoides durante a infecção por L. i. chagasi no modelo peritoneal murino. Nós observamos que a saliva aumentou a viabilidade intracelular de L. i. chagasi tanto em neutrófilos como em neutrófilos recrutados para a cavidade peritoneal. As células recrutadas para cavidade peritoneal apresentaram maiores níveis da relação PGE2/LTB4 e o pré-tratamento com NS-398 reverteu o efeito da saliva sobre a viabilidade intracelular dos parasitas. Parasitas como Leishmania são capazes de produzir PGs utilizando uma maquinaria enzimática própria. Neste estudo nós descrevemos a dinâmica de formação e a distribuição celular dos CLs em L. i. chagasi bem como a participação desta organela na produção de PGs. A quantidade de CLs aumentou durante a metaciclogênese assim como a expressão de PGF2α sintase (PGFS), sendo esta enzima co-localizada nos CLs. A adição de ácido araquidônico AA à cultura de L. i. chagasi aumentou a quantidade de CLs por parasita, bem como a secreção de PGF2α. A infecção com as diferentes formas de L. i. chagasi não foi capaz de estimular a formação de CLs na célula hospedeira. Por outro lado, os parasitas intracelulares apresentaram maiores quantidades de CLs. A infecção estimulou uma rápida expressão de COX-2, mas não foi detectado aumento na produção de PGF2α nos sobrenadantes. Por fim, nós verificamos a presença do receptor de PGF2α (FP) nos vacúolos parasitóforos de macrófagos infectados com L. i. chagasi. O prétratamento das células com um antagonista do receptor FP inibiu os índices de infecção de forma dose-dependente. Em conjunto, nossos dados apontam que os eicosanoides desempenham um papel crucial para evasão da resposta imune durante os momentos iniciais da infecção por L. i. chagasi com diferentes contribuições do parasita, do vetor e da célula hospedeira neste contexto.

Diffuse Cutaneous Leishmaniasis (LCD) is a rare clinical manifestation of Leishmaniasis, characterized by a number of macrophages heavily parasitized and low inflammatory reaction. In Brazil, Leishmania (Leishmania) amazonensis is the main specie involved in LCD cases. It has been described that the exposure and recognition of phosphatidylserine (PS) on the surface of apoptotic cells phagocytosed by macrophages is a macrophage deactivation mechanism dependent on TGF-pi and PGE2 (Fadok et al. 1998). Morover, it was demonstrated by Barcinski and colleagues that L. amazonensis amastigotes expose PS on its surface, in a mechanism called ’’Apoptotic Mimicry." In this context, our goal was to investigate the exposure of PS on the surface of L. amazonensis isolates obtained from LCD patients and its role during the infection of macrophages. Initially, peritoneal macrophages from FI mice (BALB/c x C57BL/6) stimulated with thioglycolate were infected with different L. amazonensis strains isolated from patients with Localized Cutaneous Leishmaniasis (LCL) or LCD. The exposure of PS on the surface of amastigotes was determined by flow cytometry using staining to annexin V and propidium iodide. Isolates from LCD patients showed higher PS exposure than the isolates from LCL patients 24 hours after infection. Then, we evaluated whether the differences of PS exposure in amastigotes would correlate with the infectivity of different isolates. Percentage of infected macrophages and infection index were higher in cultures using amastigotes from LCD patients compared to the ones infected with amastigotes from LCL cases. Furthermore, cultures infected with LCD isolates showed no difference to the LCL isolates regarding TGF>pl and nitric oxide production, suggesting that other immuneregulatory mechanisms are involved in this process...

Lutzomyia longipalpis saliva drives apoptosis and enhances parasite burden in neutrophils.

Neutrophils are considered the host's first line of defense against infections and have been implicated in the immunopathogenesis of Leishmaniasis. Leishmania parasites are inoculated alongside vectors' saliva, which is a rich source of pharmacologically active substances that interfere with host immune response. In the present study, we tested the hypothesis that salivary components from Lutzomyia longipalpis, an important vector of visceral Leishmaniasis, enhance neutrophil apoptosis. Murine inflammatory peritoneal neutrophils cultured in the presence of SGS presented increased surface expression of FasL and underwent caspase-dependent and FasL-mediated apoptosis. This proapoptosis effect of SGS on neutrophils was abrogated by pretreatment with protease as well as preincubation with antisaliva antibodies. Furthermore, in the presence of Leishmania chagasi, SGS also increased apoptosis on neutrophils and increased PGE(2) release and decreased ROS production by neutrophils, while enhancing parasite viability inside these cells. The increased parasite burden was abrogated by treatment with z-VAD, a pan caspase inhibitor, and NS-398, a COX-2 inhibitor. In the presence of SGS, Leishmania-infected neutrophils produced higher levels of MCP-1 and attracted a high number of macrophages by chemotaxis in vitro assays. Both of these events were abrogated by pretreatment of neutrophils with bindarit, an inhibitor of CCL2/MCP-1 expression. Taken together, our data support the hypothesis that vector salivary proteins trigger caspase-dependent and FasL-mediated apoptosis, thereby favoring Leishmania survival inside neutrophils, which may represent an important mechanism for the establishment of Leishmania infection.

Lutzomyia longipalpis saliva triggers lipid body formation and prostaglandin E2 production in murine macrophages.

BACKGROUND: Sand fly saliva contains molecules that modify the host's 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 host's inflammatory response.