Leishmania donovani-Induced Prostaglandin E2 Generation Is Critically Dependent on Host Toll-Like Receptor 2–Cytosolic Phospholipase A2 Signaling

Visceral leishmaniasis (VL) is the second-largest parasitic killer disease after malaria. During VL, the protozoanLeishmania donovaniinduces prostaglandin E2 (PGE2) generation within host macrophages to aid parasite survival. PGE2 significantly influences leishmanial pathogenesis, asL. donovaniproliferation is known to be attenuated in PGE2-inhibited macrophages. Here, we report for the first time that signaling via macrophage Toll-like receptor 2 (TLR2) plays an instrumental role in inducing PGE2 release fromL. donovani-infected macrophages. This signaling cascade, mediated via the TLR2–phosphatidylinositol 3-kinase (PI3K)–phospholipase C (PLC) signaling pathway, was found to be indispensable for activation of two major enzymes required for PGE2 generation: cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (Cox2). Inhibition of cPLA2, but not secreted phospholipase A2 (sPLA2) or calcium-independent phospholipase A2 (iPLA2), arrestedL. donovaniinfection. During infection, cPLA2 activity increased >7-fold in a calcium-dependent and extracellular signal-regulated kinase (ERK)-dependent manner, indicating that elevation of intracellular calcium and ERK-mediated phosphorylation was necessary forL. donovani-induced cPLA2 activation. For transcriptional upregulation of cyclooxygenase 2, activation of the calcium-calcineurin-nuclear factor of activated T cells (NFAT) signaling was required in addition to the TLR2-PI3K-PLC pathway. Detailed studies by site-directed mutagenesis of potential NFAT binding sites and chromatin immunoprecipitation (ChIP) analysis revealed that the binding of macrophage NFATc2, at the −73/−77 site on thecox2promoter, inducedL. donovani-drivencox2transcriptional activation. Collectively, these findings highlight the contribution of TLR2 downstream signaling toward activation of cPLA2 and Cox2 and illustrate how the TLR2-PI3K-PLC pathway acts in a concerted manner with calcium-calcineurin-NFATc2 signaling to modulate PGE2 release fromL. donovani-infected macrophages.

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Janus Kinase 3 Activity Is Necessary for Phosphorylation of Cytosolic Phospholipase A2 and Prostaglandin E2 Synthesis by Macrophages Infected with Francisella tularensis Live Vaccine Strain

Infection and Immunity ◽

10.1128/iai.01461-13 ◽

2013 ◽

Vol 82 (3) ◽

pp. 970-982 ◽

Cited By ~ 12

Author(s):

Ashley M. Brummett ◽

Aaron R. Navratil ◽

Joshua D. Bryan ◽

Matthew D. Woolard

Keyword(s):

Prostaglandin E2 ◽

Phospholipase A2 ◽

P38 Mapk ◽

Francisella Tularensis ◽

Biosynthetic Pathway ◽

Cytosolic Phospholipase A2 ◽

Janus Kinase ◽

Content Type ◽

Cytosolic Phospholipase ◽

Cox 2

ABSTRACTFrancisella tularensis, the causative agent of tularemia, modulates the host immune response to gain a survival advantage within the host. One mechanism of immune evasion is the ability ofF. tularensisto induce the synthesis of the small lipid mediator prostaglandin E2 (PGE2), which alters the host T cell response making the host more susceptible toFrancisellagrowth. PGE2is synthesized by a tightly regulated biosynthetic pathway following stimulation. The synthesis of PGE2begins with the liberation of arachidonic acid (AA) from membrane phospholipids by cytosolic phospholipase A2 (cPLA2). AA is subsequently converted to the unstable intermediate PGH2by cyclooxygenase-2 (COX-2), and PGH2undergoes an isomerization reaction to generate PGE2. Our objective was to identifyF. tularensis-activated host signaling pathways that regulate the activity of the enzymes in the PGE2-biosynthetic pathway. In this study, we show that cPLA2, p38 mitogen-activated protein kinase (MAPK), and Janus kinase 3 (JAK3) signaling are necessary forF. tularensis-induced PGE2production. Inhibition of JAK3 activity reduced the phosphorylation of cPLA2and COX-2 protein levels. In addition, JAK3 regulates cPLA2phosphorylation independent of transcription. Moreover, p38 MAPK activity is required forF. tularensis-induced COX-2 protein synthesis, but not for the phosphorylation of cPLA2. This research highlights a unique signaling axis in which JAK3 and p38 MAPK regulate the activity of multiple enzymes of the PGE2-biosynthetic pathway in macrophages infected withF. tularensis.

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