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Leishmania donovani-Induced Prostaglandin E2 Generation Is Critically Dependent on Host Toll-Like Receptor 2–Cytosolic Phospholipase A2 Signaling Amrita Bhattacharjee, Saikat Majumder, Shibali Das, Sweta Ghosh, Satabdi Biswas, Subrata Majumdar Division of Molecular Medicine, Bose Institute, Kolkata, India

Visceral leishmaniasis (VL) is the second-largest parasitic killer disease after malaria. During VL, the protozoan Leishmania donovani induces prostaglandin E2 (PGE2) generation within host macrophages to aid parasite survival. PGE2 significantly influences leishmanial pathogenesis, as L. donovani proliferation 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 from L. 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), arrested L. donovani infection. 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 for L. donovaniinduced 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 sitedirected mutagenesis of potential NFAT binding sites and chromatin immunoprecipitation (ChIP) analysis revealed that the binding of macrophage NFATc2, at the ⴚ73/ⴚ77 site on the cox2 promoter, induced L. donovani-driven cox2 transcriptional 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 from L. donovani-infected macrophages.

T

o survive and proliferate within their hosts, intracellular pathogens must devise strategies to evade the various host defense mechanisms targeted toward them. Several pathogens achieve immune evasion by inducing and sustaining the production of several immunosuppressive molecules within the host (1– 3). The causative agent of visceral leishmaniasis (VL), the protozoan parasite Leishmania donovani, is known to restrict the host defense responses by induction of various immunosuppressive compounds (4). One such compound, prostaglandin E2 (PGE2), is an arachidonic acid derivative which acts as a lipid signaling mediator (5). PGE2 is extremely versatile (6, 7) and is known to influence several key phenomena that determine the character of the immune response (8). PGE2 can impair the microbicidal capacity of macrophages (9), and it is reported that by selectively inhibiting Th1 cytokines, PGE2 induces a Th2 bias in mitogenically stimulated T cells (10). This potent Th1 inhibition has been demonstrated to prevent inflammation-induced tissue damage during gastroduodenal disease and hepatic inflammation (11, 12). However, during infection with intracellular pathogens (such as L. donovani), PGE2 can promote disease progression. Human monocytes have exhibited increased PGE2 production following Leishmania infection (13). The importance of PGE2 in the progression of VL is critical, as disease progression is arrested in PGE2-inhibited macrophages (14). This study aimed to elucidate the signaling phenomena responsible for L. donovani-induced PGE2 generation in an attempt to understand which components can be modulated to block its immunosuppressive effects. Here, we report for the first time how L. donovani infection

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initiates a host signaling cascade that compels infected macrophages to elevate PGE2 production. This signaling is accomplished with the help of a pattern recognition receptor of the Tolllike receptor (TLR) family, TLR2. Our findings reveal that signaling via TLR2–phosphatidylinositol 3-kinase (PI3K)–phospholipase C (PLC) induces the activation of cytoplasmic phospholipase A2 (PLA2) and cyclooxygenase 2 (Cox2), two key enzymes required for PGE2 synthesis. Phospholipases hydrolyze membrane phospholipids into free fatty acids and are classified into phospholipase A1, phospholipase A2, phospholipase B, phospholipase C, and phospholipase D categories, depending upon the position of the bond cleaved (15). PLA2s are further classified into secreted phospholipase A2 (sPLA2), cytosolic phospholipase A2 (cPLA2), and calcium-independent phospholipase A2 (iPLA2) (16). In this study, we determined that cPLA2, but not sPLA2 or iPLA2, regulated PGE2 generation during L. donovani infection.

Received 25 June 2016 Returned for modification 14 July 2016 Accepted 24 July 2016 Accepted manuscript posted online 1 August 2016 Citation Bhattacharjee A, Majumder S, Das S, Ghosh S, Biswas S, Majumdar S. 2016. Leishmania donovani-induced prostaglandin E2 generation is critically dependent on host Toll-like receptor 2– cytosolic phospholipase A2 signaling. Infect Immun 84:2963–2973. doi:10.1128/IAI.00528-16. Editor: J. A. Appleton, Cornell University Address correspondence to Subrata Majumdar, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Cyclooxygenases are enzymes that catalyze the metabolism of arachidonic acid for the production of PGE2. The inducible isoform, Cox2, is known to be elevated during L. donovani infection (13, 17). Our experiments determined that the TLR2-PI3K-PLC pathway is required for L. donovani-mediated transcriptional upregulation of cox2. In addition, L. donovani infection-induced cox2 transcription is critically dependent on the calcium-calcineurin-NFATc2 pathway. Collectively, these findings outline the novel roles of TLR2, PI3K, PLC, extracellular signal-regulated kinase (ERK) (for cPLA2 activation), and the calcium-calcineurinNFATc2 pathway (for cox2 transcription) in the modulation of PGE2 release from L. donovani-infected macrophages. MATERIALS AND METHODS Animals. BALB/c mice (4 to 6 weeks old) were purchased from the National Centre for Laboratory Animal Sciences (Hyderabad, India) and reared in the institute’s animal facilities. For in vitro experiments, peritoneal macrophages (PM␾s) were isolated from thioglycolated mice as described previously (18). For some experiments, bone marrow macrophages were used by incubating bone marrow-derived stem cells with 10 ng/ml of macrophage colony-stimulating factor (M-CSF) for 5 days and selecting the F4/80expressing population (19). Murine splenocyte isolation was done by perfusing the spleen with ice-cold perfusion buffer (phosphate-buffered saline [PBS], 0.5 mM EDTA, 5 mM glucose). Perfused spleen samples were cut into small pieces and homogenized. The spleen homogenate was subjected to centrifugation on Histopaque 1077 (1,400 rpm for 30 min at room temperature; Sigma), washed, and resuspended in complete culture medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum [FCS], 2 mM sodium pyruvate, 1 mM L-glutamine, 0.5 mM ␤-mercaptoethanol [␤-ME], 100 U/ml of penicillin, and 100 mg/ml of streptomycin). Studies were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council (20). All experimental animal protocols received prior approval from the Institutional Animal Ethical Committee (Bose Institute, registration number 1796/PO/Ere/S/14/CPCSEA). Infection. L. donovani strain AG83 (MHOM/IN/1983/AG83) was used for infection. Promastigotes were maintained at 22°C in M199 (supplemented with 10% FCS and 1% glutamine). Macrophages were infected with L. donovani promastigotes at a 1:10 (macrophage/parasite) ratio for the desired times, after which the unbound parasites were washed off. Intracellular parasite numbers were determined by Giemsa staining. For in vivo experiments, animals were infected by intravenous inoculation (via tail vein) of 107 L. donovani organisms resuspended in 0.1 ml of PBS (pH 7.2) (47). Mice were sacrificed 14 days after infection. Organ parasite burdens were determined by stamp-smear method. ELISA. cPLA2 activity assays were conducted using a cytosolic phospholipase A2 assay kit (Abcam) according to the manufacturer’s instructions. In some experiments, resting peritoneal macrophages were stimulated with various TLR ligands [1 ␮g/ml of PAM3CSK4 for TLR1/2, 10 ␮g/ml of PGN for TLR2/2, 30 ␮g/ml of poly(I·C) for TLR3, 10 ng/ml of lipopolysaccharide (LPS) for TLR4, 10 ng/ml of flagellin for TLR5, 3 ␮g/ml of FSL-1 for TLR2/6, 100 ng/ml of imiquimod for TLR7/8, and 10 ␮g/ml of CpG-ODN for TLR9], and cPLA2 activity was estimated. PGE2 levels were estimated using a PGE2 enzyme-linked immunosorbent assay (ELISA) kit by following the manufacturer’s instructions (Abcam, Cambridge, MA). Briefly, macrophages were incubated in the presence of 40 ␮M varespladib (sPLA2 inhibitor), 10 ␮M arachidonyl trifluoromethyl ketone (ATK; cPLA2 inhibitor), or 10 ␮M bromoenollactone (BEL; iPLA2 inhibitor) and infected with Leishmania in a 1:10 (macrophage/parasite) ratio. Cell supernatants were then harvested for PGE2 estimation. For in vivo cytokine assays, splenocytes were isolated from different experimental animal groups and incubated in the presence of soluble Leishmania antigen (SLA; 5 mg/ml) for 24 h, and culture supernatant levels of inter-

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leukin 12 (IL-12), gamma interferon (IFN-␥), tumor necrosis factor alpha (TNF-␣), IL-10, IL-4, and transforming growth factor ␤ (TGF-␤) were estimated using a sandwich ELISA kit (BD OptEIA ELISA kit; BD Biosciences, Franklin Lakes, NJ) according to the manufacturer’s instructions. siRNA synthesis. TLR2, TLR3, and TLR4-specific small interfering RNAs (siRNAs) were synthesized as previously described (21). We used the silencer siRNA construction kit (Ambion) and the following sequences: for TLR2, 5=-AAAGAGAAAGTACTTACTGCACCTGTCTC-3= (forward) and 5=-AATGCAGTAAGTACTTTCTCTCCTGTCTC-3= (reverse); for TLR3, 5=-GGAUAGGUGCCUUUCGA-3= (forward) and 5=-UGACGAAAGGCAC CUAUGC-3= (reverse); and for TLR4, 5=-AATCTATGCAGGGATTCAAGC CCTGTCTC-3= (forward) and 5=-AACATGCATTGGTAGGTAATACCTG TCTC-3= (reverse). Intracellular calcium estimation. Calcium was estimated as described previously (22). Briefly, macrophages were scraped from cell culture dishes at 1, 3, and 6 h following infection (along with uninfected controls) in chilled PBS and centrifuged at 2,000 rpm. Cell pellets were dissolved in calcium-free buffer (2 ml of 1 M HEPES, 0.5 ml of 20% glucose, 20 ml of Hanks balanced salt solution [HBSS]) and enumerated by a hemocytometer before calcium estimation. Three microliters of Fura-2/AM (3 ␮l of 1.5 mM Fura-2/AM, 2 ␮l of 20% Pluronic F-127, 10 ␮l of 10% bovine serum albumin [BSA]) was added to 1 ml of cell solution at 37°C for 30 min and transferred in quartz cuvette. mRNA quantification. Total RNAs were extracted using TRIzol reagent (Sigma). For cDNA synthesis, 1 ␮g of total RNA from each sample was reverse transcribed using RevertAid reverse transcriptase (Thermo Fisher Scientific). cDNA from each sample was amplified with 0.5 U of Taq DNA polymerase (Fermentas) in a 50-ml reaction volume under the following conditions: initial activation (2 min at 95°C) and cycling (denaturation for 30 s at 94°C, annealing for 30 s at 58°C, and extension for 1 min at 72°C for 35 cycles), using a PerkinElmer Gen Amp PCR system 2400. Sequences of the PCR primers are listed in Table 1. PCR amplified products were subsequently subjected to gel electrophoresis on a 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the housekeeping control. The respective protein/amplified cDNA bands were analyzed using a model GS-700 imaging densitometer and molecular analyst (version 1.5; Bio-Rad Laboratories, Hercules, CA). Western blotting. Macrophages from various experimental sets were scraped and centrifuged at 2,000 rpm for 15 min at 4°C. The cells were then resuspended in ice-cold extraction buffer containing 50 mM TrisHCl (pH 7.5), 50 mM EGTA, 50 mM ␤-mercaptoethanol, and protease inhibitor cocktail (Sigma). In some cases, differentially treated cells were fractionated to obtain nuclear and cytosolic fractions (23) Proteins were quantified with Bio-Rad protein assay reagent using BSA as a standard. Equal amounts of protein (50 ␮g) in each lane were subjected to sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis (SDS–10% PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked overnight with 3% bovine serum albumin in Tris-saline buffer (pH 7.5), and immunoblotting was performed (23) for detection of cPLA2 and ␤-actin expression. Reporter assay. Luciferase activity in cell extracts from infected or treated PM␾s was measured using a dual-luciferase reporter kit (Promega) according to the manufacturer’s protocols, and the luciferase activity was normalized to the protein content. ChIP assay. Chromatin immunoprecipitation (ChIP) was conducted by incubating nuclear extracts of control and infected macrophages with antibodies (Abs) for NFATc1 to -c4 isoforms for 18 h at 4°C. Total DNA was then isolated, and the ⫺73/⫺77 region of the cox2 promoter was amplified using primers listed in Table 1. Statistical variation. Each experiment was repeated at least thrice, and data are represented as means ⫾ standard deviations. Student’s t test was employed to assess the significance of the differences between the means of different groups. For some experiments, data representative of those

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TABLE 1 Sequences of primers used in this study Primer

Sequencesa

cox2

Fwd, 5=-GGAGAGACTATCAAGATAGTGATC-3= Rev, 5=-ATGGTCAGTAGACTTTTACAGCTC-3=

cox2 promoter probe

Fwd, 5=-TTCGTCTCTCATTTGCGTGGGTAA-3= Rev, 5=-CATTAGCAGCCAGTTGTCAACTGCGA-3=

SDM primers For NFAT binding site 1

Fwd, 5=-GGAAAGCCTAAGCTTAAAGACAGAGTC-3= Rev, 5=-GACTCTGTCTTTAAGCTTAGGCTTTCC-3= Fwd, 5=-GGTGGGGGTTGGTTAAAGCCTAAGC-3= Rev, 5=-GCTTAGGCTTTAACCAACCCCCACC-3= Fwd, 5=-GAAGCAGAGAGGGTTAAAAGTTGGTGG-3= Rev, 5=-CCACCAACTTTTAACCCTCTCTGCTTC-3= Fwd, 5=-ACTGGGAGGTTACCGGAGACCCCAAA-3= Rev, 5=-TGACCCTCCAATGGCTCTCTGGGGTTT-3=

For NFAT binding site 2 For NFAT binding site 3 For NFAT binding site 4 a

Fwd, forward; Rev, reverse.

from three independent experiments are provided. For in vivo experiments, four mice were used per group, and two-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for statistical assessment of significance between groups. Data are represented as means ⫾ standard errors. A P value of ⬍0.05 was considered significant, and a value of ⬍0.001 was considered highly significant.

RESULTS

Activation of macrophage cPLA2 is critical for L. donovani-induced PGE2 release. Synthesis of PGE2 requires mobilization of arachidonic acid from the membrane to the cytoplasm (24), which is accomplished by members of the phospholipase A2 (PLA2) family. This may involve sPLA2, cPLA2, or iPLA2 (25). To determine which enzyme(s) contributes to L. donovani-induced PGE2 generation, PGE2 concentrations were estimated in infected macrophages pretreated with specific inhibitors of sPLA2, cPLA2, or iPLA2 (Fig. 1A). While sPLA2 or iPLA2 inhibition resulted in PGE2 levels comparable with those of untreated infected controls, inhibition of cPLA2 caused a significant decrease in the PGE2 levels, suggesting that L. donovani-induced PGE2 was generated via cPLA2 and not by sPLA2/iPLA2 activation. Interestingly, cPLA2-inhibited macrophages were also significantly less susceptible to L. donovani infection than macrophages treated with sPLA2 or iPLA2 inhibitors (Fig. 1B). To ensure that L. donovanidriven PGE2 occurs in other macrophage types as well, we compared levels of PGE2 generation (during infection and during cPLA2 inhibition) in peritoneal and bone marrow-derived macrophages from BALB/c and C57BL/6 backgrounds (Fig. 1C). Following infection, PGE2 production was significantly elevated in both types of macrophages from both mouse backgrounds. Further studies were conducted with peritoneal macrophages from BALB/c mice. On analyzing cPLA2 enzyme activity at different time points following infection (Fig. 1D), cPLA2 activity was found to be rapidly elevated and peaked at 3 h postinfection (p.i.). Membrane translocation data also revealed greater membrane accumulation of cPLA2 in infected macrophages (versus control macrophages), especially during early infection (Fig. 1D). For validation of our in vitro findings, we conducted cPLA2 inhibition experiments in vivo. L. donovani infection of mice treated with ATK, a cPLA2 inhibitor, culminated in significantly lower splenic and hepatic

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parasite burdens (95.4% and 92.43%, respectively) than in untreated infected mice (Fig. 1E). Further, inhibition of cPLA2 resulted in increased production of the Th1 cytokines TNF-␣ and IFN-␥ from splenocytes of infected mice compared to that in infected macrophages without inhibitor (Fig. 1G). In addition, cPLA2 inhibition impaired the infection-induced upregulation of signature Th2 cytokines such as IL-10 or TGF-␤ (Fig. 1H). Interestingly, cPLA2 inhibition also resulted in increased antigen-specific T cell proliferation compared to that in uninhibited infected mice (Fig. 1F), highlighting the role of cPLA2 in suppression of host T cell responses during VL. Collectively, these findings clearly demonstrate the importance of cPLA2 in modulating various immunopathological processes during experimental VL in vitro and in vivo. Macrophage TLR2 signaling is indispensable for cPLA2 activation during L. donovani infection. To determine how cPLA2 activation was being induced, we verified whether L. donovanidriven cPLA2 activation was induced by host-parasite interaction at the cell surface (prior to internalization) or during events following intramacrophagic internalization. Leishmania-induced cPLA2 activation was found to be phagocytosis independent, as macrophages treated with the phagocytosis blocker cytochalasin D had unaltered cPLA2 activity (Fig. 2A). Interestingly, Leishmania-induced cPLA2 enzyme activity was attenuated in macrophages treated with the lipid raft disruptor ␤-methylcyclodextrin (␤-MCD). Taken together, these findings clearly indicated that an extracellular host-parasite interaction was responsible for initiating the cPLA2 signaling cascade during L. donovani infection. Previously, the extracellular pattern recognition receptor TLR2 has been reported to activate cPLA2 (26). Moreover, the Leishmania surface molecule lipophosphoglycan (LPG) can interact with the macrophage-expressed TLR2 to modulate macrophage effector functions (27). To determine whether TLR signaling contributes to cPLA2 activation during infection, cPLA2 activity was analyzed in macrophages that had been stimulated with various TLR ligands (Fig. 2B). Stimulation with ligands of TLR1/2, TLR2/2, TLR3, and TLR4 led to a significant increase in cPLA2 activation in resting macrophages. To check the role of these TLRs on cPLA2 activation during L. donovani infection, we used siRNAs against

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FIG 1 L. donovani infection activates macrophage cPLA2. (A) PM␾s were isolated from thioglycolated BALB/c mice. After 48 h to allow adherence, PM␾s were incubated with 25 ␮M varespladib (sPLA2 inhibitor) for 1 h, 10 ␮M ATK (cPLA2 inhibitor) 1 h, or 10 ␮M BEL (iPLA2 inhibitor) for 10 min and then infected with L. donovani in a 1:10 (macrophage/parasite) ratio. At different time points of infection, cell supernatants were collected, centrifuged to remove any cellular entities, and transferred to fresh tubes for PGE2 estimation by ELISA. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant reduction (**, P ⬍ 0.001) in PGE2 levels compared with uninhibited infected sets. (B) Similarly inhibited PM␾s were infected with L. donovani (1:10 ratio). After 24 h, intracellular parasite numbers were determined by Giemsa staining. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant reduction (**, P ⬍ 0.001) in cPLA2 inhibited sets compared with infected uninhibited sets. (C) Macrophages, both peritoneal (PM␾) and bone marrow derived (BMM␾), were isolated from BALB/c and C57BL/6 mice and were infected with L. donovani in vitro as described before, in the presence or absence of the cPLA2 inhibitor ATK. After 24 h, cell supernatants were collected and PGE2 levels were estimated by

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FIG 2 L. donovani-induced cPLA2 is TLR2 dependent. (A) PM␾s were incubated with 1 ␮g/ml of the phagocytosis inhibitor cytochalasin D (Cyt D), or a 5 mM

concentration of the lipid raft disruptor ␤-methylcyclodextrin (␤-MCD), and then infected with L. donovani. Three hours p.i., unbound parasites were washed off and cPLA2 activity was determined from cell lysates using the Abcam cytosolic phospholipase A2 assay kit according to the manufacturer’s instructions. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant reduction (**, P ⬍ 0.001) of cPLA2 activity in ␤-MCD-treated infected macrophages compared with the value for infected untreated macrophages. (B) Macrophages were stimulated with various TLR ligands (see Materials and Methods), and cPLA2 activity was estimated as described before. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant increase (**, P ⬍ 0.001) of cPLA2 activity in stimulated macrophages compared with the value for unstimulated macrophages. (C) PM␾s which had been treated with TLR2 siRNA, TLR3 siRNA, and TLR4 siRNA were infected with L. donovani as mentioned before. Cell lysates were then collected and used for cPLA2 activity analysis as before. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant reduction (**, P ⬍ 0.001) of cPLA2 activity in TLR2-inhibited sets compared with that in infected uninhibited sets. Next, cPLA2 activity (D) and PGE2 generation (E) were estimated as before in control (PBS), L. donovani-infected (Inf), or TLR2 agonist-treated (uninfected) macrophages. Data are the means ⫾ standard deviations from triplicate experiments.

TLR2, -3, and -4 and observed cPLA2 activity. As TLR1 signals via TLR2, we did not use TLR1 siRNA. Interestingly, blockade of TLR2 (but not TLR3 or TLR4) significantly inhibited cPLA2 activity following infection, indicating TLR2 involvement in the activation of cPLA2 in infected macrophages (Fig. 2C).

Moreover, a comparison of infected macrophages with TLR2stimulated uninfected macrophages revealed comparable levels of cPLA2 activation (Fig. 2D) and PGE2 generation (Fig. 2E). This led us to speculate that the binding of leishmanial LPG to TLR2 on the macrophage cell surface is the initial necessary

ELISA. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant reduction (*, P ⬍ 0.05; **, P ⬍ 0.001) in cPLA2-inhibited sets compared with infected uninhibited sets. (D) For cPLA2 activity, PM␾s were infected with L. donovani as before. At different time points, unbound parasites were washed thrice with sterile PBS and were lysed for protein isolation. This cell lysate was used for activity analysis using the Abcam cytosolic phospholipase A2 assay kit according to the manufacturer’s instructions. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant (**, P ⬍ 0.001) increase in cPLA2 activity at 3 h p.i. compared to the value at 0 h of infection. For cPLA2 membrane isolation, PM␾s were infected with L. donovani as before. Three hours p.i., cells were harvested, and membrane (memb) and cytoplasmic (cyt) fractions were extracted for protein isolation and subjected to immunoblotting for detection of cPLA2. The blot shown is a representative of those from experiments performed in triplicate, yielding similar observations. (E to H) In vivo effect on cPLA2 inhibition. (E) Mice were intravenously infected with L. donovani as before, and the cPLA2 inhibitor ATK (7.5 mg/kg) was administered by oral gavage daily. After 30 days, the parasite burdens in the liver and spleen were determined by the stamp-smear method and expressed as Leishman Donovan units (LDU). Untreated infected mice were used as controls. Data are the means ⫾ standard errors for four mice per group. Asterisks indicate a statistically significant reduction (*, P ⫽ 0.001) of parasite burden compared with that of untreated infected mice. (F) In vitro proliferation of spleen cells of differently treated mice was determined 30 days postinfection. Proliferative responses to 5 ␮g/ml of SLA by splenocytes from L. donovani-infected mice were studied in the presence or absence of cPLA2 inhibition. Proliferation was measured by [3H]thymidine incorpofration. Data are means ⫾ standard errors from triplicate experiments yielding similar results. Asterisks indicate a statistically significant increase (**, P ⬍ 0.001) of T cell proliferation compared with value for infected mice (in counts per minute). Splenocytes were isolated from mice 30 days postinfection and stimulated with 5 ␮g/ml of SLA for 24 h. The spleen cell culture supernatants were then analyzed to study the status of the Th1 cytokines IL-12, IFN-␥, and TNF-␣ (G) and the Th2 cytokines IL-10, IL-4, and TGF-␤ (H) by ELISA. Results are expressed as means ⫾ standard errors of data from four replicate experiments. Asterisks indicate statistically significant (*, P ⬍ 0.05; **, P ⬍ 0.001) alterations in cytokine levels of PLA2-inhibited infected mice compared to values for uninhibited infected mice.

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FIG 3 L. donovani induces macrophage cPLA2 in a PI3K-PLC-Ca2⫹-ERK-dependent manner. (A) PM␾s were pretreated with wortmannin (PI3K inhibitor) and U-73122 (PLC inhibitor) and infected with L. donovani. Three hours p.i., unbound parasites were washed off and cPLA2 activity was determined from cell lysates as before. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant reduction (**, P ⬍ 0.001) of cPLA2 activity following infection in PI3K-inhibited and PLC-inhibited macrophages compared with the value for infected uninhibited macrophages. (B) Uninfected and L. donovani-infected PM␾s were scraped and centrifuged, and the pellet was dissolved in calcium-free buffer (2 ml of 1 M HEPES, 0.5 ml of 20% glucose, 20 ml of HBSS). PM␾s were enumerated by hemocytometer and incubated with 3 ␮l of Fura-2/AM (3 ␮l of 1.5 mM Fura-2/AM, 2 ␮l of 20% Pluronic F-127, 10 ␮l of 10% BSA) added in 1 ml of cell solution at 37°C for 30 min. Cells were centrifuged and the pellets were dissolved in calcium-free buffer and transferred in a quartz cuvette. Mean fluorescence intensity was monitored at different time intervals by flow cytometry. Data acquisition was performed for 300 s, and after every 10 s, the intracellular calcium concentration was plotted. Data points on the graph represent these 10-s intervals. Data are representative of one of three different experiments conducted under identical conditions. (Inset) Effect of EDTA on intracellular calcium mobilization induced by L. donovani. Macrophages were treated with EDTA or left untreated and then infected with L. donovani for 3 h and treated with Fura-2/AM (5 ␮M). Mean fluorescence intensity was estimated by flow cytometry. Data are the means ⫾ standard deviations from triplicate experiments. (C) PM␾s were incubated in the presence of a 10 ␮M concentration of the cell-permeative Ca2⫹ chelator BAPTA and infected with L. donovani. cPLA2 activity was determined from cell lysates as before. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant reduction (**, P ⬍ 0.001) of cPLA2 activity in BAPTA-treated infected PM␾s compared to that of untreated infected PM␾s. (D) PM␾s were preincubated with 100 ␮mol/ml of PD98059 (ERK inhibitor), 10 ␮g/ml of SB203580 (p38 inhibitor), or 10 ␮M SP600125 (JNK inhibitor) and infected with L. donovani. cPLA2 activity was determined from cell lysates as before. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant reduction (**, P ⬍ 0.001) of cPLA2 activity in ERK-inhibited macrophages compared with that in infected uninhibited macrophages.

step in L. donovani-induced cPLA2 activation and consequent PGE2 generation. cPLA2 activation occurs in a PI3K-PLC-calcium- and ERKdependent manner. To determine whether TLR2 directly interacted with cPLA2, coimmunoprecipitation studies were performed, but no direct interaction was observed (data not shown), indicating that other signaling intermediates were relaying the activation signal downstream of TLR2. cPLA2 membrane docking requires calcium (28), and, in epithelial cells, TLR2 ligand binding is known to activate calcium signaling in a PI3K-PLC-dependent manner (29). Therefore, we wanted to check whether PI3K-PLC signaling could influence the activation of cPLA2 during L. donovani infection by using specific inhibitors of PI3K and PLC (Fig. 3A). Inhibition of both PI3K and PLC significantly restricted cPLA2 activity. Also, to study how L. donovani-induced cPLA2 was influenced by infection-induced alterations in calcium concentrations, calcium was quantified at different time points of infection (Fig. 3B). L. donovani-infected macrophages had ele-

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vated calcium levels, and the highest calcium levels were obtained around 3 h postinfection. For analyzing whether cPLA2 activation was mediated by calcium derived from extracellular sources or by mobilization of intracellular calcium, macrophages were treated with either cell-impermeative EDTA (to chelate extracellular divalent cations) or cell-permeative intracellular calcium ion chelator BAPTA-AM. Infection of EDTA-treated and untreated macrophages had comparable intracellular calcium levels (Fig. 3B, inset). BAPTA-AM-treated macrophages failed to activate cPLA2 during L. donovani infection, whereas EDTA treatment had no effect (Fig. 3C). These findings confirm the role of intracellular calcium alterations in Leishmania-induced cPLA2 activation and indicate that cPLA2 activation proceeds via a calcium-driven, PI3K-driven, and PLC-dependent manner. Following calcium-induced membrane translocation of cPLA2, a mitogen-activated protein kinase (MAPK)-mediated phosphorylation event allows the catalytic subunit of cPLA2 to be exposed to the membrane (30). To identify which MAPKs were responsible

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FIG 4 L. donovani induced cox2 and PGE2 under TLR2-PI3K-PLC-PLA2 inhibition conditions. (A) PM␾s were infected with L. donovani (1:10 ratio). At various time points of infection, unbound parasites were washed off, and cells were collected in TRIzol reagent for mRNA isolation. cDNA synthesis and reverse transcription-PCR (RT-PCR) of the isolated mRNA were performed to study transcription of cox2 and GAPDH expression. Data are representative of those from three independent experiments yielding similar observations. (B) PM␾s were pretreated with TLR2 siRNA, wortmannin (PI3K inhibitor), and U-73122 (PLC inhibitor) or pyrrophenone (cPLA2 inhibitor) and infected with L. donovani. Twenty-four hours later, mRNA isolation was done as before, and RT-PCR of the isolated mRNA was performed to study transcription of cox2 and GAPDH expression. Data are representative of those from three independent experiments yielding similar observations. (C) In another experiment, similarly inhibited PM␾s were infected with L. donovani, and PGE2 generation was assayed from the cell supernatant. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant inhibition (**, P ⬍ 0.001) of PGE2 in variously inhibited macrophages compared with that in infected uninhibited macrophages.

for infection-induced cPLA2 activation, macrophages pretreated with different MAPK inhibitors were infected, and cPLA2 enzyme activity was studied. While cPLA2 activity was unaffected by p38MAPK or Jun N-terminal protein kinase (JNK) inhibition, ERK inhibition impaired cPLA2 activity (Fig. 3D), highlighting the role of ERK in the activation of cPLA2 during L. donovani infection. Together, these results prove that L. donovani-induced cPLA2 requires activation of the TLR2-PI3K-PLC pathway along with ERK phosphorylation for its activity. Role of TLR2-PI3K-PLC signaling in L. donovani-induced cox2 upregulation. It is now well established that L. donovani infection induces PGE2 release from infected macrophages (13, 14), which involves upregulation of Cox2. Our lab has previously reported increased Cox2 expression during L. donovani infection (17). Semiquantitative PCR studies at various time points of infection validated that L. donovani-infected macrophages upregulated cox2 mRNA as early as 1 h p.i. and sustained this elevation until 24 h (Fig. 4A). To determine whether cox2 activation was modulated by the cPLA2 activation pathway induced by L. donovani, cox2 expression was analyzed under TLR2, PI3K, PLC, and PLA2 inhibition conditions. Inhibition of TLR2, PI3K, and PLC significantly impaired L. donovani-induced cox2 upregulation (Fig. 4B), underlining the importance of TLR2-PI3K-PLC signaling. To verify that this signaling was responsible for regulating

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PGE2 release from infected macrophages, we estimated PGE2 concentrations from supernatants of TLR2-, PI3K-, and PLC-inhibited macrophages (Fig. 4C). PGE2 production was severely diminished under TLR2, PI3K, and PLC inhibition conditions. Together, these findings demonstrate that L. donovani-induced cox2 upregulation and PGE2 synthesis were TLR2-PI3K-PLC dependent. In addition, cox2 transcription, but not PGE2 production, was cPLA2 independent. L. donovani-induced cox2 activation in a calcium-calcineurin-NFATc2-dependent manner. Since infection-induced Cox2 was TLR2-PI3K-PLC dependent, the effect of calcium on Leishmania-induced cox2 expression was determined. Depletion of intracellular calcium completely arrested cox2 upregulation during infection at both the mRNA (Fig. 5A) and protein (Fig. 5B) levels, highlighting the importance of calcium in L. donovani-induced cox2 transcriptional activation. In this context, the role of calcineurin, a calcium-sensitive serine-threonine phosphatase reported to influence Cox2 expression, was analyzed. Inhibition of calcineurin with FK-506, a specific inhibitor, significantly abrogated L. donovani-driven cox2 expression (Fig. 5B). These findings prompted us to study whether cox2 upregulation following infection was mediated by NFAT, a calcineurin-dependent transcription factor. cox2 transcription was monitored in the presence or absence of an NFAT-inhibitory peptide, VIVIT. We found that

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FIG 5 Role of the Ca2⫹-calcineurin-NFAT pathway in L. donovan-induced cox2 transcription. PM␾s were incubated in the presence of 10 ␮M BAPTA (a

cell-permeative Ca2⫹ chelator) (A), 10 ␮M FK-506 (a calcineurin inhibitor) (B), and 100 ␮M VIVIT (an NFAT-inhibitory peptide) (C) and infected with L. donovani. Twenty-four hours later, mRNA isolation, cDNA synthesis, and RT-PCR were performed to study transcription of cox2 and GAPDH expression. Data are representative of those from three independent experiments yielding similar observations. Data represent the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant reduction (**, P ⬍ 0.001) of cPLA2 activity in inhibited PM␾s compared to that in uninhibited infected controls. (D) Schematic representation of murine cox2 promoter with putative NFAT binding sites. Numbers indicate distance in base pairs from the transcription start site. (E) Analysis of cox2 promoter deletion constructs: luciferase-tagged plasmids (10 ␮g/35-mm dish) containing different promoter regions of the cox2 promoter (as shown on the left) were transfected into RAW264.7 cells. The macrophages were then infected with L. donovani for 4 h and assayed for luciferase activity. The luciferase activity was measured and expressed as a percentage of the luciferase activity of the whole promoter construct (⫺963/⫹70). Data are the averages from three independent experiments. (F) Luciferase activity of lysate of RAW264.7 cells transfected with either whole-length cox2 promoter construct (⫺963/⫹70) or deletion constructs of the cox2 promoter (lacking site 1, site 2, site 3, or site 4), followed by infection with L. donovani. The luciferase activity was measured and expressed as a percentage of the ⫺963/⫹70 construct, defined as 100. Data are the averages from three independent experiments. (G) L. donovani activates NFATc2 to bind with the cox2 promoter. ChIP analysis of cox2 promoter (⫺134/⫹70) with nuclear extract derived from macrophages infected with L. donovani and assessed with Abs to NFATc1, NFATc2, NFATc3, and NFATc4, followed by semiquantitative PCR amplification using primers specific to the cox2 promoter.

VIVIT-treated macrophages weakly induced cox2 in response to leishmanial challenge (Fig. 5C), illustrating the critical role of NFAT in L. donovani-induced cox2 transcription. Promoter analysis of the murine cox2 promoter revealed four potential (5=-GGAAA-3=) NFAT binding sequences at positions ⫺77/⫺73 (site 1), ⫺90/⫺86 (site 2), ⫺111/⫺107 (site 3), and ⫺314/⫺310 (site 4) (Fig. 5D). To determine which site(s) was required for L. donovani-driven cox2 transcription, we performed transfection experiments using truncated variants of the cox2 promoter luciferase construct. Following Leishmania infection, macrophages transfected with promoter constructs lacking the site 4 sequence (truncated at ⫺203) exhibited luciferase activity similar to that of macrophages transfected with constructs carrying the full-length cox2 promoter (⫺963 to ⫹70 bp), proving that the sequence at ⫺314/⫺310 kb had no significant role in L. donovaniinduced-NFAT-driven cox2 transcription (Fig. 5E). In contrast, macrophages transfected with constructs lacking sites 1, 2, and 3 demonstrated significantly diminished luciferase activity following infection, indicating that one or more of the sequences at regions ⫺77/⫺73, ⫺90/⫺86, and ⫺111/⫺107 were required for

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NFAT-mediated transcriptional activation of the cox2 gene during Leishmania infection. Since these three potential sites lay in very close proximity, we performed site-directed mutagenesis at each sequence to identify those necessary for Leishmania-induced cox2 transcription. In stably transfected and infected macrophages, mutation of site 1 significantly reduced the luciferase activity under the cox2 promoter compared to results with the wild-type (WT) promoter construct, signifying the importance of this NFAT binding site in cox2 transcription during Leishmania infection (Fig. 5F). However, mutation of site 2, 3, or 4 exerted no significant alterations in luciferase activity (compared to nonmutated promoter constructs), suggesting little or no importance of these sites during Leishmania-induced cox2 activation. Among the five known NFAT isoforms, NFATc1 to -c4 are activated by the calcium-calcineurin pathway, while NFAT5 is unresponsive to calcium (31). Since cox2 expression was already observed to be calcium dependent (Fig. 5A), we focused on NFATc1 to -c4. To identify which NFAT isoform was important for regulation of cox2 transcription during Leishmania infection, we performed ChIP assay using primers spanning ⫺135/⫺4 bp of the

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cox2 promoter region in the presence of specific antibodies against the NFATc1, NFATc2, NFATc3, and NFATc4 isoforms. Our results indicated that out of the various calcium-responsive NFAT isoforms (NFATc1 to -c4), NFATc2 was majorly responsible for cox2 transcriptional activation during Leishmania infection (Fig. 5G). DISCUSSION

The most abundant prostanoid in the body, PGE2, can influence disease outcome by promoting an immunosuppressive environment. A growing body of evidence highlights the importance of this lipid mediator in the immunopathology of cancer (5–7) and infectious diseases caused by protozoa, viruses, and bacteria (4, 32–35). Several species of Leishmania are reported to upregulate macrophage PGE2 production and to exploit the immunosuppressive properties of PGE2 to survive within the hostile intramacrophagic environment (13, 36, 37). However, the signaling events that stimulate PGE2 release following L. donovani infection were unclear. In this study, we have delineated the signaling pathways that drive PGE2 synthesis in L. donovani-infected macrophages, and we have uncovered the importance of TLR2 signaling in this process. First, we observed that Leishmania infection increased macrophage cPLA2 activity (Fig. 1D) and that this activation of cPLA2 (but not sPLA2 or iPLA2) was crucial for PGE2 release following L. donovani infection (Fig. 1A). cPLA2-inhibited macrophages had significantly reduced parasite counts, underlining the importance of this enzyme in promoting leishmanial pathogenesis (Fig. 1B). In our in vivo experiments as well, cPLA2 inhibition was observed to restrict spleen and liver parasite burdens by 95.4% and 92.43%, respectively (Fig. 1E). Moreover, by studying cytokine generation from splenocytes of cPLA2-inhibited and infected mice, we found that cPLA2 inhibition significantly upregulated the proinflammatory cytokines IL-12, IFN-␥, and TNF-␣ (Fig. 1G) and suppressed the anti-inflammatory cytokines IL-10, IL-4, and TGF-␤ (Fig. 1H). Further, to validate the effect of various components of the PGE2 inducing pathway, we analyzed cytokine levels (the IL-10/IL-12 ratio) in infected macrophages during TLR2, PI3K, PLC, calcium, ERK1/2, calcineurin, and NFAT inhibition (Fig. 6). Our data revealed cytokine modulation similar to that with cPLA2 inhibitors. These findings demonstrate how L. donovani-induced cPLA2 activation modulates the cytokine milieu in favor of a Th2 response. L. donovani is known to alter TLR2 downstream signaling (38). In our study, we observed that Leishmania-induced cPLA2 activation was TLR2 dependent (Fig. 2B), thus revealing yet another role of TLR2 in facilitating leishmanial pathogenesis. Surprisingly, while TLR2, TLR3, and TLR4 stimulation of resting macrophages resulted in cPLA2 activation, TLR3 or TLR4 silencing exerted no effect on cPLA2 activity in infected macrophages (Fig. 2B). These phenomena indicate that although stimulation via other TLRs can activate cPLA2 in resting macrophages, cPLA2 signaling in infected macrophages occurs in a TLR2-dependent manner. In this context, it is reasonable to speculate that leishmanial surface protein(s), such as LPG, may preferentially interact with macrophage TLR2 during early infection, thus triggering the signaling cascade. In agreement with a previous report (13), we also found insignificant changes in cPLA2 mRNA during infection (data not shown), suggesting that the increased cPLA2 activity observed following infection was mediated by posttranscriptional changes in

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FIG 6 Cytokine modulation during inhibition of components of the PGE2 signaling pathway. Macrophages were treated with specific inhibitors of TLR, PI3K, PLC, calcium signaling, ERK1/2, calcineurin, and NFAT as before and then infected with L. donovani at a1:10 ratio. After 24 h of infection, cell supernatants were collected for ELISA. Data are the means ⫾ standard deviations from triplicate experiments. Asterisks indicate a statistically significant alteration (**, P ⬍ 0.001) in IL-10 versus IL-12 ratio in inhibited infected PM␾s compared to that in the uninhibited infected control.

the protein (calcium binding, phosphorylation). Very few reports have addressed the changes in calcium homeostasis during L. donovani infection (4, 22). Calcium binding is reported to promote the affinity of cPLA2 for membrane PIP2-phosphate moieties (39). We analyzed the kinetics of calcium levels during the early time points of L. donovani infection and observed maximum calcium elevation at 3 h postinfection (Fig. 3B). This was also the time at which we recorded highest cPLA2 activity and membrane accumulation (Fig. 1D). Additionally, chelation of intracellular calcium at this time point (3 h) significantly attenuated cPLA2 activity (Fig. 3C), suggesting that L. donovani-induced intracellular calcium elevation was mandatory for cPLA2 activation. This report underscores the impact of calcium signaling in L. donovaniinduced PGE2 synthesis, as blocking the calcium signal simultaneously impaired cPLA2 activation and transcription of cox2, another enzyme indispensable for PGE2 generation. After calcium binding-induced cPLA2 membrane attachment, further phosphorylation of the catalytic domain by an MAPK enables the cPLA2 active site to be exposed, thereby permitting physical interaction of the catalytic domain with substrate phospholipids (40). Prior reports have already established ERK as an important modulator of Leishmanial pathogenesis (41, 42). Here, we report that ERK plays a crucial role as a cPLA2 activator as well, as ERK-inhibited infected macrophages exhibited 80% less cPLA2 activity than uninhibited infected macrophages (Fig. 3D). Next, we elucidated the signaling events that lead to cox2 transcription during VL. Interestingly, inhibition of cPLA2 had no effect on cox2 expression (Fig. 4). This occurrence led us to speculate that a nonlinear signaling pathway was involved in activation of cPLA2 and Cox2. We hypothesize that this point of divergence is marked by the elevation of intracellular calcium, as elevation of calcium ions was necessary for both cPLA2 activation (by direct

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FIG 7 Proposed model for L. donovani-mediated host TLR2 downstream signaling that activates cPLA2 and Cox2, culminating in PGE2 generation. TLR2 stimulation by a leishmanial protein (possibly LPG) activates signaling via PI3K and PLC. This results in an elevation of intracellular calcium levels, which, along with ERK phosphorylation, leads to activation of cPLA2. This increase in calcium levels also activates the calcium-calcineurin-NFAT pathway and specifically induces NFATc2 binding to the ⫺73/⫺77 NFAT binding site on the cox2 promoter, activating cox2 transcription.

binding) (Fig. 4C) and cox2 transcriptional activation (by calcineurin-NFATc2) (Fig. 5A). L. donovani-driven cox2 upregulation was regulated by calcineurin, a calcium-sensitive serine-threonine phosphatase (Fig. 5B). To our knowledge, this is the first study regarding the role of macrophage calcineurin during leishmanial infection. Moreover, the calcineurin-dependent transcription factor NFAT was necessary for cox2 induction (Fig. 5C to F), as NFAT-inhibited macrophages failed to induce cox2 postinfection. Out of the four putative NFAT binding sites, site 1 at ⫺77/⫺73 was observed to be the most important sequence for NFAT-dependent transcription of cox2, as site-directed mutagenesis of site 1 (but not site 2, 3, or 4) resulted in a significant decrease of cox2 transcription following infection (Fig. 5F). Recently, it has been shown that different isoforms of NFAT are activated in response to distinct subcellular calcium signaling events (43). In our study, NFATc2 (also known as NFAT1) was found to be the isoform that mediates L. donovanimediated cox2 transcription (Fig. 5G). Taken together, our findings illustrate that L. donovani-mediated PGE2 generation is a complex process involving several intermediates (Fig. 7). Inhibition of PGE2 production has previously been proposed as a therapeutic alternative in the treatment of several diseases (13, 17, 32, 44–46). In this regard, this study provides several targets and reveals their hitherto-unknown roles. The critical cues provided by this report can fuel the development of new molecules/approaches aimed at the ultimate reduction of the PGE2-mediated immunosuppressive state of the host. ACKNOWLEDGMENTS Mouse cox2 promoter reporter plasmid constructs were generous gifts from Susan Fischer (M. D. Anderson Cancer Center, Texas). We are also grateful to the director of Bose Institute for providing space and other

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instrumental facilities. We acknowledge Prabal Gupta and the Central Instrument Facility, Bose Institute, for technical assistance. We also thank the University Grants Commission (UGC), Government of India, for providing a fellowship to Amrita Bhattacharjee.

FUNDING INFORMATION This work, including the efforts of Amrita Bhattacharjee, was funded by University Grants Commission (UGC).

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