Prostaglandins from Cytosolic Phospholipase A2α/Cyclooxygenase-1 Pathway and Mitogen-activated Protein Kinases Regulate Gene Expression in Candida albicans-infected Macrophages*

In Candida albicans-infected resident peritoneal macrophages, activation of group IVA cytosolic phospholipase A2 (cPLA2α) by calcium- and mitogen-activated protein kinases triggers the rapid production of prostaglandins I2 and E2 through cyclooxygenase (COX)-1 and regulates gene expression by increasing cAMP. In C. albicans-infected cPLA2α−/− or COX-1−/− macrophages, expression of Il10, Nr4a2, and Ptgs2 was lower, and expression of Tnfα was higher, than in wild type macrophages. Expression was reconstituted with 8-bromo-cAMP, the PKA activator 6-benzoyl-cAMP, and agonists for prostaglandin receptors IP, EP2, and EP4 in infected but not uninfected cPLA2α−/− or COX-1−/− macrophages. In C. albicans-infected cPLA2α+/+ macrophages, COX-2 expression was blocked by IP, EP2, and EP4 receptor antagonists, indicating a role for both prostaglandin I2 and E2. Activation of ERKs and p38, but not JNKs, by C. albicans acted synergistically with prostaglandins to induce expression of Il10, Nr4a2, and Ptgs2. Tnfα expression required activation of ERKs and p38 but was suppressed by cAMP. Results using cAMP analogues that activate PKA or Epacs suggested that cAMP regulates gene expression through PKA. However, phosphorylation of cAMP-response element-binding protein (CREB), the cAMP-regulated transcription factor involved in Il10, Nr4a2, Ptgs2, and Tnfα expression, was not mediated by cAMP/PKA because it was similar in C. albicans-infected wild type and cPLA2α−/− or COX-1−/− macrophages. CREB phosphorylation was blocked by p38 inhibitors and induced by the p38 activator anisomycin but not by the PKA activator 6-benzoyl-cAMP. Therefore, MAPK activation in C. albicans-infected macrophages plays a dual role by promoting the cPLA2α/prostaglandin/cAMP/PKA pathway and CREB phosphorylation that coordinately regulate immediate early gene expression.

C. albicans Culture-C. albicans (ATCC 10261) was grown on Sabouraud dextrose agar plates and maintained at 4°C. The day before the experiment, it was streaked onto a fresh plate and incubated overnight at 37°C. C. albicans was scraped from the plate and washed twice in endotoxin-free PBS. Live C. albicans at a multiplicity of infection of 2-5 was used. C. albicans expressing green fluorescent protein (GFP) was kindly provided by Dr. Robert Wheeler (University of Maine). It was generated from the wild type SC5314 strain and exhibits similar virulence as the wild type strain (27). C. albicans strains 10261 and SC5314 are both serotype A and have similar effects in RPM.
RPM Isolation-RPM were obtained by peritoneal lavage as described previously (14). Cells were plated at a density of 5 ϫ 10 5 cells/cm 2 (48-well plate) and incubated for 2 h at 37°C in a humidified atmosphere of 5% CO 2 in air. After washing the cultures to remove non-adherent cells, the adherent macrophages were incubated in DMEM containing 10% heat-inactivated FBS, 100 g/ml streptomycin sulfate, 100 units/ml penicillin G, and 0.29 mg/ml glutamine for 16 -18 h at 37°C. The cells were washed twice with serum-free DMEM containing 0.1% BSA (stimulation medium) and then infected with C. albicans.
Mass Spectrometry Eicosanoid Analysis-The culture medium was collected after macrophage stimulation, centrifuged, and then stored at Ϫ80°C. The media were thawed and mixed with an equal volume of cold methanol prior to analysis. The samples were diluted in water to a final methanol concentration of Ͻ15% and then extracted using a solid phase extraction cartridge (Strata Polymeric Reversed Phase, 60 mg/ml, Phenomenex, Torrance, CA). Metabolites were separated by HPLC that directly interfaced into the electrospray ionization source of a triple quadrapole mass spectrometer (Sciex API 3000, PE-Sciex, Thornhill, Canada). Mass spectrometric analyses were performed in the negative ion mode using multiple-reaction monitoring for specific analytes as described previously (24).
RT-PCR-RNA was isolated from RPM and treated with DNase to remove contaminating genomic DNA, and cDNA was generated using 0.5 g of RNA. PCRs contained, in a final volume of 20 l, 10 l of 2ϫ TaqMan fast universal master mix, 1 l of 20ϫ TaqMan assay/probe, and 9 l of cDNA (30 -70 ng) in RNase-free water. The Thermal Fast cycle program was as follows: 20 s at 95°C followed by 40 cycles of 1 s at 95°C and 20 s at 60°C, using the StepOne Plus RT-PCR system (Applied Biosystems). Duplicate reactions were analyzed for each sample. The following TaqMan assay probes were used: Gusb Mm01197698_ml; Nr4a2 Mm00443060_ml; Il10 Mm00439614_ml; Tnf Mm99999068_ml; Ptgs2 Mm00478364_ml. Threshold cycle values (C T ) were determined and used to calculate relative gene expression using 2 Ϫ⌬CT analysis (28). For normalization of gene expression, the C T values of the gene of interest were related to the C T values of the housekeeping gene Gusb, which is expressed at a constant level in treated and untreated RPM. A relative expression value of 1 indicates that the gene of interest is expressed at the same level as Gusb.
Western Blotting-Cell monolayers were washed twice in icecold PBS and then lysed in a modified radioimmune precipitation assay buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 1 mM EGTA, 1 mM EDTA, protease and phosphatase inhibitors. After incubation on ice for 30 min, lysates were centrifuged at 15,000 rpm for 15 min, and protein concentration in the supernatant was determined. Lysates were boiled for 5 min after the addition of Laemmli sample buffer, and then proteins were separated on 10% SDS-polyacrylamide gels. After transfer to nitrocellulose membrane, samples were incubated in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween with 5% nonfat milk (blocking buffer) for 1 h and then incubated overnight at 4°C with primary antibodies in blocking buffer. The membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000) in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween for 1 h at room temperature. The immunoreactive proteins were detected using the Amersham Biosciences ECL system (GE Healthcare).
Macrophage Function Assays-For evaluating binding and internalization (recognition assay) of GFP-C. albicans, cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM (1 ϫ 10 5 ) were seeded onto MatTek 35-mm dishes and incubated for 2 h. After washing, cells were incubated overnight followed by infection with live GFP-C. albicans (multiplicity of infection of 2) in phenol red-free stimulation medium for 30 min at 37°C and 5% CO 2 . Macrophages were washed, fixed with 4% paraformaldehyde for 15 min, and then stained with DAPI. Images were captured on a Marianas 200 spinning disk confocal microscope using Intelligent Imaging Innovation Inc. software (Slidebook version 6.0) to determine the number of macrophages containing GFP-C. albicans.
For determining C. albicans killing, cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM were plated as described above and then incubated with and without IFN␥ (200 units/ml) overnight, followed by infection with C. albicans (multiplicity of infection of 2) in stimulation medium with and without IFN␥ (200 units/ ml) for 4 h. After removing the culture medium, 1% Triton X-100 was added, which lyses RPM but not C. albicans. C. albicans viability was measured using the XTT cell viability kit as described (29). The procedure measures the ability of viable cells to convert XTT to formazan by enzymes that are inactivated in dead cells. Wells containing an equivalent number of C. albicans without RPM were included as a positive control for determining 100% viability.
For measuring nitric oxide production, RPM were cultured as described for the killing assay. After a 4-h incubation of RPM with C. albicans, culture media were removed, centrifuged, and used to measure nitric oxide production using the Griess reagent. To measure NADPH oxidase activity, superoxide anion production was determined by measuring cytochrome c reduction as described previously (20).
Statistics-Statistics were calculated in GraphPad using unpaired t test to obtain two-tailed p values.

Role of COX-1-derived Prostaglandins in Regulating Gene
Expression-cPLA 2 ␣ initiates the release of arachidonic acid for production of prostaglandins and leukotrienes in RPM within minutes of infection with C. albicans (24). Prostaglandin I 2 (PGI 2 ) and prostaglandin E 2 (PGE 2 ) are produced before the expression of COX-2, suggesting that they are derived from the COX-1 pathway. By comparing C. albicans-infected cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM, we previously found that cPLA 2 ␣-mediated production of prostaglandins regulates gene expression, including immediate early genes, such as Nr4a2, that are expressed within the first 30 -60 min of infection (24). To specifically investigate the role of COX-1-derived prostaglandins in regulating gene expression, RPM from wild type and COX-1 Ϫ/Ϫ mice were compared. C. albicans induced rapid production of PGE 2 and 6-keto-PGF 1 ␣, the stable metabolite of PGI 2 , in COX-1 ϩ/ϩ RPM, but their levels were reduced to base-  line in COX-1 Ϫ/Ϫ RPM infected for up to 3 h (Fig. 1a). In contrast, there was no significant difference in the release of arachidonic acid and leukotriene C 4 production. cPLA 2 ␣-mediated prostaglandin production in response to C. albicans influences expression of genes to dampen inflammation highlighted by an increase in the anti-inflammatory cytokine IL-10 and suppression of the pro-inflammatory cytokine TNF␣ (24). The expression of these genes and the immediate early gene Nr4a2 was compared in C. albicans-infected COX-1 ϩ/ϩ and COX-1 Ϫ/Ϫ RPM (Fig. 1b). Expression of Il10 and Nr4a2 was significantly lower, and Tnf␣ expression was higher, in COX-1 Ϫ/Ϫ compared with COX-1 ϩ/ϩ RPM. Expression of Tnf␣ in COX-1 Ϫ/Ϫ RPM increased from 1-6 h after C. albicans infection, whereas expression of Nr4a2 and Il10 in COX-1 ϩ/ϩ RPM was transient, peaking at 1 and 3 h, respectively.
COX-2 Expression in C. albicans-infected RPM Requires COX-1-derived Prostaglandins-Although results show a role for COX-1-derived prostaglandins in regulating gene expression, most genes that were higher in cPLA 2 ␣ ϩ/ϩ RPM compared with cPLA 2 ␣ Ϫ/Ϫ RPM peaked 3 h after C. albicans infection (24). Because COX-2 protein can be detected by 3 h in C. albicans-infected cPLA 2 ␣ ϩ/ϩ RPM, prostaglandins derived from COX-2 may contribute to regulating gene expression. COX-1 Ϫ/Ϫ RPM are a useful model to investigate a role for COX-2, but it was important to confirm that COX-2 was expressed at similar levels in C. albicans-infected COX-1 ϩ/ϩ and COX-1 Ϫ/Ϫ RPM. In some tissues/cells, COX isoforms compensate for each other when one is ablated (30). Conversely, prostaglandins either added exogenously or produced endogenously can promote COX-2 expression via cAMP-response elements (31)(32)(33)(34). In COX-1 ϩ/ϩ RPM, COX-2 was detected on Western blots at 3 h and at higher levels by 6 h but was not detected or was extremely low in COX-1 Ϫ/Ϫ RPM (Fig.  2a). Expression of Ptgs2 mRNA correlated with the Western blotting data showing higher levels in COX-1 ϩ/ϩ than in COX-1 Ϫ/Ϫ RPM after C. albicans infection (Fig. 2b). The results suggest that prostaglandins derived from COX-1 transcriptionally regulate COX-2 expression by increasing cAMP. Consistent with this possibility, COX-2 expression was increased by treating COX-1 Ϫ/Ϫ RPM with 8-Br-cAMP and by agonists for the PGI 2 receptor IP (iloprost) and PGE 2 receptors EP2 (butaprost) and EP4 (CAY10598) (Fig. 2c). The receptor agonists and 8-Br-cAMP did not increase COX-2 expression in uninfected COX-1 Ϫ/Ϫ RPM, suggesting that cAMP acts synergistically with additional signals triggered by C. albicans.
We also confirmed that cPLA 2 ␣ activation regulates expression of COX-2, which was lower in C. albicans-infected cPLA 2 ␣ Ϫ/Ϫ RPM than in cPLA 2 ␣ ϩ/ϩ at 3 and 6 h after stimulation (Fig. 3a). As observed in COX-1 Ϫ/Ϫ RPM, the expression of COX-2 was increased by treating cPLA 2 ␣ Ϫ/Ϫ RPM with 8-Br-cAMP, iloprost, and butaprost (Fig. 3b). Treatment with the EP4 receptor agonist (CAY10598) also increased COX-2 expression in cPLA 2 ␣ Ϫ/Ϫ RPM, as observed in COX-1 Ϫ/Ϫ RPM (data not shown). The expression of COX-2 in C. albicans-infected cPLA 2 ␣ ϩ/ϩ RPM was blocked by the IP receptor antagonist CAY10441, suggesting a role for PGI 2 (Fig. 3b). We also investigated the role of PGE 2 in regulating COX-2 expression by using antagonists for EP2 and EP4 receptors. It was necessary to evaluate different concentrations of the antagonists because their IC 50 concentrations differ. At the higher concentration of the receptor antagonists tested, they all blocked COX-2 expression in C. albicans-infected cPLA 2 ␣ ϩ/ϩ RPM, suggesting a lack of specificity at this concentration ( Fig. 3c). At the lower concentration tested, the receptor antagonists partially blocked COX-2 expression, and this allowed us to evaluate their combined effects (Fig. 3c). When C. albicans-infected cPLA 2 ␣ ϩ/ϩ RPM were treated with a combination of two antagonists at the lower concentration, there was partial inhibition of COX-2 expression, but when all three were tested, complete inhibition was observed (Fig. 3c, right). The results suggest that endogenous production of both PGI 2 and PGE 2 acting through the IP, EP2, and EP4 receptors all contribute to regulating expression of COX-2.
We also investigated the role of prostaglandin receptors in regulating gene expression by using agonists for IP (iloprost), EP2 (Ptgs2), and EP4 (CAY10598). The agonists increased expression of COX-2 (Ptgs2), Nr4a2, and Il10 mRNA and decreased Tnf␣ expression in C. albicans-infected cPLA 2 ␣ Ϫ/Ϫ RPM but not in uninfected RPM except for a small increase in Nr4a2 expression (Fig. 4). The IP, EP2, and EP4 agonists were equally effective in regulating expression of Nr4a2, Ptgs2, and Tnf␣ when tested alone or in combination. However, Il10 expression was increased to a greater extent by iloprost and butaprost than with the EP4 agonist CAY10598 (Fig. 4). When CAY10598 was tested in combination with IP and EP2 agonists, expression of Il10 was significantly lower than with iloprost and butaprost alone, suggesting that a distinct signaling pathway through EP4 dampens Il10 expression.

Role of MAPKs in Regulating Gene
Expression-Our next approach was to identify the signals triggered by C. albicans that act synergistically with prostaglandin-induced production of cAMP for regulating expression of Ptgs2, Nr4a2, and Il10. We focused on the role of MAPKs because they are implicated as important regulators of these immediate early genes, although there are cell type-dependent differences in their action (35)(36)(37)(38). We previously reported that C. albicans and zymosan stimulate activation of ERK1/2, p38, and JNK1/2 in RPM (15,20). We first compared ERK1/2 and p38 activation (evaluated using phospho-specific antibodies) in cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM to determine whether their activation was influenced by the cPLA 2 ␣/prostaglandin/cAMP pathway (Fig. 5a). ERKs and p38 were rapidly activated in both cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM in response to C. albicans. The extent of p38 activation was similar in cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM and remained elevated from 15 to 60 min. Activation of ERKs, which was slightly lower in cPLA 2 ␣ Ϫ/Ϫ RPM, peaked at 15 min and then decreased by 60 min. In some cells, cAMP promotes p38 activation, but our results demonstrate that cPLA 2 ␣-mediated prostaglandin production has no effect on p38 activation (39,40).
To determine whether activation of MAPKs in response to C. albicans infection regulates gene expression, we tested the effect of inhibitors that block activation of ERKs (U0126) and p38 (SB20219). ERKs and p38 regulate cPLA 2 ␣ activation; therefore, the MAPK inhibitors were not tested in cPLA 2 ␣ ϩ/ϩ RPM because they would inhibit cPLA 2 ␣-mediated arachidonic acid release, prostaglandin production, and increases in cAMP. We used C. albicans-infected cPLA 2 ␣ Ϫ/Ϫ RPM treated with and without the IP receptor agonist, iloprost, to investigate the contribution of cAMP induced by iloprost and activation of MAPK in regulating gene expression (Fig. 5b). In C. albicansinfected cPLA 2 ␣ Ϫ/Ϫ RPM, the increase in expression of Ptgs2, Nr4a2, and Il10 induced by iloprost was significantly inhibited by SB20219 together with U0126. When tested alone, U0126 and SB20219 partially decreased expression of Nr4a2. Expression of Ptgs2 and Il10 was inhibited by SB20219 but not by U0126. The results show that both ERKs and p38 contribute to Nr4a2 expression, but p38 and not ERKs regulate Il10 and Ptgs2. In contrast, increases in cAMP and signals from C. albicans have opposing effects on Tnf␣ expression, which is lower in cPLA 2 ␣ Ϫ/Ϫ RPM treated with iloprost due to inhibition by cAMP (Fig. 5b). The higher level of Tnf␣ expressed in C. albicans-infected cPLA 2 ␣ Ϫ/Ϫ RPM not treated with iloprost was significantly blocked by U0126 together with SB20219, but neither alone had any effect. The results demonstrate that prostaglandins act synergistically with MAPK activation to induce expression of Ptgs2, Nr4a2, and Il10 in response to C. albicans but suppress expression of Tnf␣, which is dependent on both ERKs and p38. To provide further support that MAPKs act together with prostaglandins to regulate gene expression, we tested structurally distinct inhibitors of ERKs (SCH772984) and p38 (LY2228820) in C. albicans-infected COX1 Ϫ/Ϫ RPM (Fig.  5c). These inhibitors had effects very similar to those of U0126 and SB20219 on gene expression, and the results additionally show that the effects are consistent in cPLA 2 ␣ Ϫ/Ϫ and COX1 Ϫ/Ϫ RPM. Experiments were also carried out to deter-mine whether there was a correlation between the effects of MAPK inhibitors on gene expression and levels of COX2 and NR4A2 protein (Fig. 5d). In cPLA 2 ␣ Ϫ/Ϫ RPM, C. albicans and iloprost synergistically induced expression of COX2 and NR4A2 protein, but neither alone was effective. Protein expression of COX-2 and NR4A2 was blocked by inhibition of ERKs and p38, confirming that mRNA expression correlates with protein levels.
Role of JNK1 and JNK2 in Regulating Gene Expression and Eicosanoid Production-In addition to ERKs and p38, JNKs are also implicated in regulating gene expression, including Ptgs2 (38,41). As shown in Fig. 6a, C. albicans stimulated activation of JNK1 and JNK2 to a similar extent in cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM, which peaked at 30 min. We first tested the effect of the JNK inhibitor SP600125, which inhibits both JNK1 and JNK2. SP600125 significantly inhibited expression of Ptgs2, Il10, and Tnf␣ in C. albicans-infected cPLA 2 ␣ ϩ/ϩ RPM and in cPLA 2 ␣ Ϫ/Ϫ RPM treated with and without iloprost (Fig. 6b). In contrast, expression of Nr4a2 was increased by SP600125 (Fig.  6b). Western blotting showed that C. albicans induced an increase in NR4A2 protein in cPLA 2 ␣ ϩ/ϩ RPM, and expression was enhanced by the SP600125 inhibitor, consistent with the effect on mRNA levels (Fig. 6c). In contrast, SP600125 inhibited the increase in COX-2 protein in C. albicans-infected cPLA 2 ␣ ϩ/ϩ RPM. Expression of NR4A2 and COX2 was not detected in cPLA 2 ␣ Ϫ/Ϫ RPM treated with or without the SP600125. A role for JNK in activating cPLA 2 ␣ has been suggested from studies using SP600125 (42,43). We tested the effect of SP600125 concentration on gene expression and eicosanoid production (Fig. 7). SP600125 dose-dependently blocked release of arachidonic acid and production of prostaglandins and leukotriene C 4 (Fig. 7a). Similar concentrations of SP600125 increased expression of Nr4a2 and decreased expression of Ptgs2, Il10, and Tnf␣ (Fig. 7b). Although the results suggest that JNKs may play a role in regulating gene expression and cPLA 2 ␣ activation in C. albicans-infected RPM, SP600125 inhibits several other kinases with equal potency (44,45). In light of this, we tested the effect of SP600125 on activation of MAPKs and found that in addition to inhibition of JNK1/2 phosphorylation, SP600125 partially inhibited activation of ERKs and p38 in C. albicans-infected cPLA 2 ␣ ϩ/ϩ RPM (Fig. 7c).
Because the effect of SP600125 on gene expression and eicosanoid production may be due to inhibition of ERKs and p38, another approach was used to investigate the role of JNKs by comparing RPM from WT and JNK1 Ϫ/Ϫ or JNK2 Ϫ/Ϫ mice. As observed with the JNK inhibitor SP600125, the expression of Nr4a2 mRNA was significantly higher in C. albicans-infected JNK2 Ϫ/Ϫ than JNK2 ϩ/ϩ RPM, but there was no difference in expression of Ptgs2, Il10, or Tnf␣ (Fig. 8a). In contrast to the transient expression of Nr4a2 observed in cPLA 2 ␣ ϩ/ϩ RPM (BALB/c) and COX-1 ϩ/ϩ RPM (BL6/129) (see Fig. 1b), Nr4a2 expression in JNK2 Ϫ/Ϫ and JNK2 ϩ/ϩ RPM (C57BL/6) continued to increase from 1 to 6 h, which may reflect strain differences (Fig. 8a). The results suggest that JNK2 activation negatively regulates Nr4a2 expression and that SP600125 enhances expression of Nr4a2 by inhibition of JNK2. The release of arachidonic acid and eicosanoid production in response to C. albicans infection was not significantly different in JNK2 ϩ/ϩ and JNK2 Ϫ/Ϫ RPM with the exception of 6-keto-PGF1␣, which was slightly higher in JNK2 Ϫ/Ϫ RPM (Fig. 8b). A comparison of WT and JNK1 Ϫ/Ϫ RPM showed no differences in gene expression (Fig. 8c) or eicosanoid production (Fig. 8d). The results indicate that JNK2 negatively regulates expression of Nr4a2 but that neither JNK1 nor JNK2 plays a role in regulating expression of Ptgs2, Tnf␣, or Il10 or activation of cPLA 2 ␣ in C. albicans-infected RPM. The ability of SP600125 to inhibit gene  expression and cPLA 2 ␣ activation may be due to a nonspecific effect on ERK and p38 activation (see Fig. 7c).

Role of MAPKs and cAMP in Regulating Gene
Expression and CREB Phosphorylation-The transcription factor CREB regulates expression of immediate early genes containing cAMPresponse element sites in response to cAMP, including Ptgs2, Tnf␣, Il10, and Nr4a2 (46 -54). One mechanism for CREB activation is phosphorylation of Ser-133 that can be mediated by either PKA or by kinases downstream of MAPKs (55)(56)(57). In a previous study, we found that C. albicans stimulates greater production of cAMP in cPLA 2 ␣ ϩ/ϩ than in cPLA 2 ␣ Ϫ/Ϫ RPM, consistent with an autocrine effect of endogenously produced prostaglandins on gene expression (24). Experiments were carried out to determine whether the regulation of gene expression by the cPLA 2 ␣/prostaglandin/cAMP pathway was mediated by PKA or Epacs, the two main cAMP effectors (58). We previously reported that the PKA inhibitor H89 suppressed expression of Il10 and Nr4a2 and enhanced expression of Tnf␣ in C. albicans-infected cPLA 2 ␣ ϩ/ϩ (24). Another approach was used to determine the contribution of PKA and Epac in regulating gene expression that involved treating C. albicans-infected cPLA 2 ␣ Ϫ/Ϫ RPM with cAMP analogs that selectively activate PKA (6-Bnz-cAMP) or Epac (8-pCPT-2-O-Me-cAMP-AM). Expression of Ptgs2, Il10, and Nr4a2 was induced, and TNF␣ expression was suppressed, with 6-Bnz-cAMP and 8-Br-cAMP but not by the Epac activator (Fig. 9a). The results sug-gest that prostaglandins regulate transcription through cAMPmediated PKA activation.
Experiments were then carried out to determine whether CREB phosphorylation was downstream of the prostaglandin/ cAMP/PKA or MAPK pathways. The phosphorylation of CREB on Ser-133 occurred to a similar extent in C. albicans-infected cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM and COX-1 ϩ/ϩ and COX-1 Ϫ/Ϫ RPM (Fig. 9b). At 10 min, there was a low level of CREB phosphorylation that increased by 30 min and remained elevated for 60 min. The results demonstrate that the prostaglandin/cAMP/PKA pathway does not regulate CREB phosphorylation on Ser-133. We then determined whether CREB phosphorylation is downstream of MAPKs (Fig. 9c). In C. albicans-infected cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM, phosphorylation of CREB was blocked by the p38 inhibitor SB20219 but not by blocking ERK activation with the MEK1 inhibitor U0126. The very low level of CREB phosphorylation that remained in RPM treated with the p38 inhibitor was not evident when RPM were treated with both the ERK and p38 inhibitor, suggesting a minor role for ERKs. Similar results were obtained with the structurally distinct ERK (SCH772984) and p38 (LY2228820) inhibitors when tested in COX-1 ϩ/ϩ and COX-1 Ϫ/Ϫ RPM. The results suggest that the role of p38 in regulating gene expression is through phosphorylation of CREB.
Another approach was used to study the role of p38 MAPK in promoting CREB phosphorylation and gene expression in RPM. This involved treating RPM with anisomycin, a stimulator of cell stress that we previously found preferentially activates p38 but not ERKs in RPM (20). Studies have shown that activation of p38 by anisomycin initiates a kinase cascade leading to the phosphorylation of CREB and regulation of gene expression (59,60). Treatment of cPLA 2 ␣ Ϫ/Ϫ RPM with anisomycin activated p38 and induced phosphorylation of CREB (Fig. 10, a and b). In contrast, activation of PKA with 6-Bnz-cAMP or treatment with the IP receptor agonist iloprost did not induce phosphorylation of CREB or enhance CREB phosphorylation induced by anisomycin (Fig. 10, a  and b). The p38 inhibitor LY2228820 blocked CREB phosphorylation in cPLA 2 ␣ Ϫ/Ϫ RPM treated with anisomycin. We carried out experiments to evaluate PKA activation in cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM using an antibody that detects substrates phosphorylated by AGC family kinases, including PKA (Fig. 10c). Western blots show that the PKA activator 6-Bnz-cAMP induced phosphorylation of a 60-kDa protein in both cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM, suggesting that it is a PKA substrate (Fig. 10c). Importantly, Western blots using the phospho-(Ser/Thr) PKA antibody show that C. albicans induces phosphorylation of the 60-kDa protein in cPLA 2 ␣ ϩ/ϩ but not in cPLA 2 ␣ Ϫ/Ϫ RPM. The results suggest that C. albicans stimulates PKA activation through the cPLA 2 ␣/prostaglandin/cAMP pathway in cPLA 2 ␣ ϩ/ϩ , resulting in phosphorylation of the 60-kDa protein but that C. albicans does not activate PKA in cPLA 2 ␣ Ϫ/Ϫ RPM by a mechanism independent of cAMP. In addition, there is little phosphorylation of the 60-kDa protein in RPM treated with anisomycin, suggesting that it poorly activates PKA. The results suggest that phosphorylation of CREB is downstream of p38 activation stimulated by either C. albicans or anisomycin and not by PKA in RPM. As we observed with C. albicans, anisomycin acted synergistically with either 6-Bnz-cAMP or iloprost to induce expression of Ptgs2, Nr4a2, or IL10 but had no effect when tested alone (Fig. 10d). However, unlike C. albicans, anisomycin did not induce TNF␣ expression in cPLA 2 ␣ Ϫ/Ϫ RPM, suggesting the need for additional signaling pathways stimulated by C. albicans. Unexpectedly, TNF␣ expression was induced by anisomycin together with 6-Bnz-cAMP or iloprost but only when p38 was inhibited. The basis for these results is not known. Role of cPLA 2 ␣ in Regulating Macrophage Function-Eicosanoid production can affect innate immune responses by regulating gene expression and macrophage function, including bacterial uptake and killing (61)(62)(63). Experiments were carried out to determine the role of cPLA 2 ␣ in regulating C. albicans recognition and killing and the production of superoxide anion and nitric oxide in response to C. albicans infection. There were no differences in the ability of cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM to bind and internalize GFP-C. albicans (recognition assay) (Fig. 11a). C. albicans infection stimulated NADPH oxidase activation (superoxide anion production), which was significantly higher in cPLA 2 ␣ Ϫ/Ϫ than in cPLA 2 ␣ ϩ/ϩ RPM (Fig.  11b). In contrast, superoxide anion production in cPLA 2 ␣ Ϫ/Ϫ and cPLA 2 ␣ ϩ/ϩ RPM was not significantly different in response to PMA, a known activator of NADPH oxidase. Incubation of RPM overnight with INF␥ promoted nitric oxide production that occurred to a greater extent in uninfected RPM. In both uninfected and infected RPM, nitric oxide production was significantly higher in cPLA 2 ␣ Ϫ/Ϫ than in cPLA 2 ␣ ϩ/ϩ RPM (Fig.  11c). The lower production of nitric oxide in C. albicans-infected RPM is consistent with reports that C. albicans suppresses nitric oxide production that may be due to inhibition of nitric-oxide synthase (64,65). The killing of C. albicans, tested using an assay that measures C. albicans viability, was found to be higher in cPLA 2 ␣ Ϫ/Ϫ than in cPLA 2 ␣ ϩ/ϩ RPM, reflecting the increased production of superoxide and nitric oxide (Fig.  11d). Treating cPLA 2 ␣ ϩ/ϩ RPM with the cyclooxygenase inhibitor indomethacin enhanced their ability to kill C. albicans to the level observed in cPLA 2 ␣ Ϫ/Ϫ RPM, suggesting that killing is suppressed by prostaglandins (Fig. 11e). Prostaglandin E 2 has been reported to suppress bacterial killing in macrophages by inhibiting NADPH oxidase (66).

Discussion
The results of this study demonstrate how the rapid activation of cPLA 2 ␣ by C. albicans in RPM and production of prostaglandins from constitutively expressed COX-1 influence the expression of immediate early genes that regulate host defense and the extent of inflammation. Resident tissue macrophages are important first responders to microbial invasion that provide signals for initiating host defense mechanisms (16). Macrophages from different tissues exhibit considerable diversity and distinct properties due to unique transcriptional signatures (67). Recent studies have shown that the specialized phenotype of RPM and ability for self-renewal is determined by the unique expression of GATA6, which is not expressed in other commonly studied primary mouse macrophage populations, such as thioglycollate-elicited peritoneal macrophages and bone marrow-derived macrophages (68,69). RPM are one of the most highly studied tissue macrophage populations and have been used extensively to investigate the regulation of cPLA 2 ␣ activation and eicosanoid production, particularly in response to fungal cell walls (zymosan) (18 -20, 23, 70 -73). They express relatively high levels of COX-1, which contributes to the rapid production of prostaglandins in response to cPLA 2 ␣ activation (24,74). Compared with thioglycollate-elicited peritoneal macrophages and bone marrow-derived macrophages, RPM have higher expression of COX-1 and greater capacity for prostanoid production in response to a Toll-like receptor-4 agonist (9). The extent of cPLA 2 ␣ activation by C. albicans and the level of COX-1 expression contribute to the relatively high levels of prostaglandins produced and secreted within minutes after infection.
The ability of prostaglandins to influence host cell responses is determined by the types of specific G-protein-coupled receptors that are expressed for inducing autocrine or paracrine responses (10). RPM express PGE 2 (EP2, EP4) and PGI 2 (IP) receptors, and results using antagonists for these receptors suggest that collectively they contribute to the induction or suppression of immediate early genes by increasing cAMP. Endogenously produced PGI 2 and PGE 2 acting through these receptors suppress production of the pro-inflammatory cytokine TNF␣ and increase expression of Ptgs2, the transcription factor Nr4a2, and the anti-inflammatory cytokine Il10. These effects are reproduced by treating cPLA 2 ␣or COX-1-deficient RPM with 8-Br-cAMP. The results suggest that prostaglandins/ cAMP regulate gene expression through PKA activation because the PKA-selective (6-Bnz-cAMP) but not the Epacselective in part determined by the amounts produced and the level of the receptor. There is more PGI 2 than PGE 2 produced in C. albicans-infected RPM, and expression of IP is higher than that of EP2 or EP4 (24). However, the EP4 receptor agonist was less effective at inducing Il10 expression and, when tested together with the IP and EP2 agonists, reduced the levels of Il10 that were expressed compared with using the IP and EP2 agonists alone. Although EP2, EP4, and IP mediate increases in cAMP, there are differences in signaling pathways induced by PGE 2 through EP4 and EP2 (10,75). Differential signaling through EP2 and EP4 has opposing effects in Th17 cells, with EP4 mediating inhibition of Il10 production, as observed in RPM (76). EP4 can couple to pertussis toxin-sensitive G protein G i , leading to activation of phosphatidylinositol 3-kinase signaling that suppresses cAMP signaling (10). However, the EP4 agonist did not cause a generalized suppression of cAMP signaling in C. albicans-infected RPM because the effect was specific to Il10 and not Ptgs2 or Nr4a2. The activation of cPLA 2 ␣ globally affects transcriptional responses, but our focus on immediate early genes in this study illustrates how prostaglandins function as immune modulators to rapidly influence the balance of pro-and anti-inflammatory cytokines for moderating the extent of inflammation during infection. cPLA 2 ␣ activation and prostaglandin production serve to dampen inflammation by modulating the transcriptional response through cAMP, as exemplified by the induction of the immunosuppressive cytokine IL10 and suppression of TNF␣ production (24). The transcription factor NR4A2, which is rapidly induced in C. albicans-infected cPLA 2 ␣ ϩ/ϩ but not in cPLA 2 ␣ Ϫ/Ϫ RPM, has been shown to play a role in dampening inflammation (77,78). The feedback regulation of COX-2 expression by prostaglandins through increases in cAMP is well documented, but there are cell-specific differences in mechanisms of induction (32,52). In C. albicans-infected RPM, the rapid production of prostaglandins by COX-1 induces the expression of COX-2, but in thioglycollate-elicited macrophages, the initial induction of COX-2 in response to LPS promotes further COX-2 induction (32). This cAMP-regulated cytokine profile of C. albicans-infected cPLA 2 ␣ ϩ/ϩ RPM (increased IL10 and decreased TNF␣) and expression of COX-2 are characteristics of resolution phase macrophages that function to restore normal tissue function after injury (79). Our results demonstrate that in response to agonists, such as C. albicans, that strongly activate cPLA 2 ␣ in cells that constitutively express relatively high amounts of COX-1, such as RPM, the rapid production of prostaglandins can also regulate macrophage responses during the early phases of infection. Stimulation of pro-inflammatory responses in macrophages by microbial pathogens is important for host defense by recruiting myeloid cells to combat infection; however, negative feedback loops are important for dampening excess inflammation to avoid tissue damage (80,81). Prostaglandin production represents one of the pathways for controlling excess inflammation, but prostaglandins can also negatively influence the ability of macrophages to kill microbial pathogens, as we observed with C. albicans and as others have shown with bacteria (66). This emphasizes the importance of maintaining the balance of proand anti-inflammatory pathways for combating infection. This study focused on understanding how the production of endogenous prostaglandins initiated by cPLA 2 ␣ in response to infection regulates the expression of immediate early genes. By comparing wild type and both cPLA 2 ␣ Ϫ/Ϫ and COX-1 Ϫ/Ϫ RPM, as well as prostaglandin receptor agonists and antagonists, the results demonstrate that prostaglandins act synergistically with activation of MAPKs to regulate gene expression. JNKs are implicated in the regulation of Ptgs2 and Tnf␣ expression, but we were particularly interested in whether JNKs regulated gene expression through activation of cPLA 2 ␣, as reported in studies using the JNK inhibitor SP600125 (36,42,43,82). Our initial screen of the JNK inhibitor SP600125 showed that it inhibited eicosanoid production and gene expression; however, results from JNK1 Ϫ/Ϫ and JNK2 Ϫ/Ϫ RPM demonstrated that JNK1/2 do not regulate these processes. A thorough characterization has shown that SP600125 exhibits poor specificity (44). The ability of SP600125 to block eicosanoid production and gene expression can be explained by our finding that it blocks activation of ERKs and p38, which are required for cPLA 2 ␣ activation. This highlights the importance FIGURE 11. C. albicans killing and production of superoxide and nitric oxide are higher in cPLA 2 ␣ ؊/؊ RPM compared with cPLA 2 ␣ ؉/؉ RPM. a, the ability of cPLA 2 ␣ ϩ/ϩ (WT) and cPLA 2 ␣ Ϫ/Ϫ (KO) RPM to bind and internalize GFP-C. albicans (recognition assay) is shown. b, to measure NADPH oxidase activity, superoxide anion production by cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM infected with C. albicans or treated with PMA (320 nM) for 90 min was determined. c, nitric oxide production by cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM was measured after treatment with and without IFN␥ for 18 h, followed by infection with C. albicans for 4 h. d, for measuring C. albicans killing using the XTT viability assay, cPLA 2 ␣ ϩ/ϩ and cPLA 2 ␣ Ϫ/Ϫ RPM were treated with and without IFN␥ for 18 h and then incubated with C. albicans for 4 h. e, for measuring the effect of prostaglandins in regulating C. albicans killing, cPLA 2 ␣ ϩ/ϩ RPM were incubated with and without IFN␥ for 18 h and then pretreated with indomethacin (Indo; 10 M) for 30 min prior to incubation with C. albicans for 4 h (n ϭ 3; *, p Ͻ 0.05 compared with C. albicans-infected cPLA 2 ␣ ϩ/ϩ RPM (b), cPLA 2 ␣ ϩ/ϩ RPM treated with IFN␥ (c), cPLA 2 ␣ ϩ/ϩ RPM incubated with and without IFN␥ (d); and cPLA 2 ␣ ϩ/ϩ RPM not treated with indomethacin (e)). Error bars, S.E.
of using alternative approaches in determining a role for JNK in cPLA 2 ␣ activation (43,82). The only role for JNK that was uncovered in our studies was the finding that JNK2 is a negative regulator of Nr4a2 expression from experiments using SP600125 and JNK2 Ϫ/Ϫ RPM. JNKs play both a positive and negative role in regulating gene expression in a variety of cell types (36). In contrast to SP600125, inhibitors that block ERKs and p38 have well characterized specificity and have been important in defining the role of these MAPKs in activating cPLA 2 ␣ in RPM by phosphorylating Ser-505 (20,45,83). We could not determine the role of MAPKs in regulating gene expression independent of prostaglandin production by treating cPLA 2 ␣ ϩ/ϩ RPM with ERK and p38 inhibitors due to inhibition of cPLA 2 ␣. The comparison of RPM from WT and either cPLA 2 ␣ Ϫ/Ϫ or COX-1 Ϫ/Ϫ mice treated with and without iloprost provided a system to independently interrogate how prostaglandins and MAPKs contribute to gene expression. The regulation of immediate early gene expression is complex, involving many signal transduction pathways that are cell type-and stimulus-dependent (84,85). Increases in cAMP by agents that engage G-protein-coupled receptors, such as prostaglandins, and activation of MAPKs are commonly used mechanisms to regulate gene expression and immune responses (37,86). There is considerable cross-talk between these pathways, and increases in cAMP can promote or inhibit activation of ERKs and p38, depending on the cell type (39,40). We found that activation of p38 and ERKs is similar in C. albicans-infected wild type and COX-1 Ϫ/Ϫ and cPLA 2 ␣ Ϫ/Ϫ RPM, indicating that the cPLA 2 ␣/COX-1/prostaglandin/cAMP pathway has little effect on MAPK activation. Our data suggest that MAPKs, particularly activation of p38, contribute to regulation of gene expression by promoting CREB phosphorylation. A comparison of wild type and cPLA 2 ␣ Ϫ/Ϫ or COX-1 Ϫ/Ϫ RPM showed no differences in the extent of CREB phosphorylation, indicating that phosphorylation is not downstream of the prostaglandin/cAMP/PKA pathway. In addition, treating COX-1 Ϫ/Ϫ RPM with the PKA activator 6-Bnz-cAMP did not induce CREB phosphorylation, although 6-Bnz-cAMP acted synergistically with C. albicans to induce expression of Ptgs2, Nr4a2, and Il10 and suppress Tnf␣. Our results show that CREB phosphorylation stimulated by C. albicans infection and by the stress inducer anisomycin is downstream of p38 activation. A number of kinases downstream of p38, including MSK1/2, MK2, and ribosomal S6 kinases, can mediate CREB phosphorylation (59,60,(87)(88)(89). CREB phosphorylation that occurs downstream of p38 in response to stress signals, such as C. albicans, is not sufficient for transcription of CREB-regulated genes but requires additional co-activators (53,55,90). The cPLA 2 ␣/ prostaglandin/cAMP/PKA pathway may function synergistically with MAPK activation by regulating CREB co-activators, such as transducers of regulated CREB (also called CREB-regulated transcription co-activator), that enhance CREB-mediated transcription of cAMP-responsive genes (49,53,58,91,92).
As shown in other cell models, the expression of Tnf␣ in C. albicans-infected RPM involves activation of both ERKs and FIGURE 12. Pathways regulating induction of immediate early genes in C. albicans-infected RPM. C. albicans infection of RPM rapidly stimulates activation of MAPKs and calcium increases through MyD88-, Dectin-1-, and Dectin-2-mediated pathways. ERKs and p38 phosphorylate cPLA 2 ␣, which enhances catalytic activity, and calcium binds to the C2 domain, which promotes translocation to the Golgi and the phagosome. Arachidonic acid released by cPLA 2 ␣ is metabolized by constitutively expressed COX-1 to PGI 2 and PGE 2 . COX-1 is an integral membrane protein primarily localized in the endoplasmic reticulum but is also found in the Golgi. Binding of PGI 2 to the IP receptor and of PGE 2 to the EP2 and EP4 receptors increases cAMP and activation of PKA, which acts synergistically with p38-dependent CREB phosphorylation, resulting in induction of Ptgs2, Nr4a2, and Il10 and suppression of Tnf␣. AA, arachidonic acid.
p38, which regulate transcriptional and post-transcriptional pathways (54). The MAPK isoform p38␣ plays a major role in regulating Tnf␣ production by mediating CREB phosphorylation, as we observed in RPM, which primarily express the p38␣ isoform (24,93). cPLA 2 ␣ activation opposes MAPK-dependent expression of Tnf␣ through prostaglandin-induced increases in cAMP. The ability of cAMP-induced PKA activation to both enhance and suppress gene expression in macrophages involves the binding of PKA subtypes to different scaffold proteins (A kinase-anchoring proteins) that differentially regulate signaling pathways for gene expression (94). The cAMP-mediated suppression of TNF␣ production has been shown to involve binding of PKA to AKAP95, leading to inhibition of NFB (94).
In summary, C. albicans infection of RPM rapidly stimulates activation of MAPKs and calcium increases through MyD88-, Dectin-1-, and Dectin-2-mediated pathways (Fig. 12) (15). ERKs and p38 phosphorylate cPLA 2 ␣ that enhances catalytic activity, and calcium binds to the C2 domain that promotes translocation to the Golgi and the phagosome (20,23). In RPM, arachidonic acid released by cPLA 2 ␣ is metabolized by constitutively expressed COX-1 to PGI 2 and PGE 2 . COX-1 is an integral membrane protein primarily localized in the endoplasmic reticulum but is also found in the Golgi (95,96). Engagement of IP by PGI 2 and of EP2 and EP4 by PGE 2 increases cAMP and activation of PKA, which acts synergistically with p38-dependent CREB phosphorylation, resulting in induction of Ptgs2, Nr4a2, and Il10 and suppression of Tnf␣. These pathways are important in controlling the balance of cytokines produced early in response to infection for regulating innate immune responses and the extent of inflammation.
Author Contributions-C. C. L. conceived and coordinated the study and wrote the paper. B. Y. designed, performed, and analyzed experiments. R. C. M. contributed to the design, acquisition of data, and analysis of experiments using mass spectrometry. H. L., S. J., and S. S. provided technical assistance and contributed to the preparation of the figures. All authors reviewed the results and approved the final version of the manuscript.