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Macrophage’s Proinflammatory Response to a Mycobacterial Infection Is Dependent on Sphingosine Kinase-Mediated Activation of Phosphatidylinositol Phospholipase C, Protein Kinase C, ERK1/2, and Phosphatidylinositol 3-Kinase¹

Department of Biological Sciences, Center for Tropical Disease Research and Training, University of Notre Dame, Notre Dame, IN 46556

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Previous studies have shown that the ability of Mycobacterium tuberculosis to block a Ca²⁺ flux is an important step in its capacity to halt phagosome maturation. This affect on Ca²⁺ release results from M. tuberculosis inhibition of sphingosine kinase (SPK) activity. However, these studies did not address the potential role of SPK and Ca²⁺ in other aspects of macrophage activation including production of proinflammatory mediators. We previously showed that nonpathogenic Mycobacterium smegmatis and to a lesser extent pathogenic Mycobacterium avium, activate Ca²⁺-dependent calmodulin/calmodulin kinase and MAPK pathways in murine macrophages leading to TNF- production. However, whether SPK functions in promoting MAPK activation upon mycobacterial infection was not defined in these studies. In the present work we found that SPK is required for ERK1/2 activation in murine macrophages infected with either M. avium or M. smegmatis. Phosphoinositide-specific phospholipase C (PI-PLC) and conventional protein kinase C (cPKC) were also important for ERK1/2 activation. Moreover, there was increased activation of cPKC and PI3K in macrophages infected with M. smegmatis compared with M. avium. This cPKC and PI3K activation was dependent on SPK and PI-PLC. Finally, in macrophages infected with M. smegmatis compared with M. avium, we observed enhanced secretion of TNF-, IL-6, RANTES, and G-CSF and found production of these inflammatory mediators to be dependent on SPK, PI-PLC, cPKC, and PI3K. These studies are the first to show that the macrophage proinflammatory response following a mycobacterial infection is regulated by SPK/PI-PLC/PKC activation of ERK1/2 and PI3K pathways.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
As intramacrophage pathogens, mycobacteria have evolved multiple mechanisms to promote their entry and survival in host cells. One well-characterized modification of host macrophages by mycobacteria is the inhibition of phagosome/lysosome fusion and avoidance of the degradative environment of a phagolysosome. In recent years, this ability of pathogenic mycobacteria to inhibit phagosome maturation has been under extensive study (for reviews see Refs.1 and 2). Interestingly, one of the initial cellular events disrupted by Mycobacterium tuberculosis is the transient elevation of intracellular Ca²⁺. This calcium flux is required for the subsequent phagosome/lysosome fusion and induction of a Ca²⁺ flux upon an M. tuberculosis infection leads to phagosome maturation (3). This calcium rise, which occurs upon infection with dead M. tuberculosis or live Staphylococcus aureus, is dependent on sphingosine kinase (SPK)³ activation. In contrast, live M. tuberculosis fails to activate SPK (4). SPK is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms, which include Ca²⁺ mobilization, activation of MAPK, and vesicular trafficking (5, 6, 7, 8, 9).

Infection by mycobacteria leads to a signaling response by the host macrophage and subsequent production of proinflammatory mediators. However, our studies as well as others indicate that macrophages infected with pathogenic mycobacteria produce significantly less TNF- and other proinflammatory molecules compared with cells infected with nonpathogenic mycobacteria (10, 11). A modulation of host cell signaling responses is critical for the suppression of a generalized inflammatory response and the persistence of mycobacteria within the host (12). Macrophage signaling pathways that are differentially activated by infection with pathogenic compared with nonpathogenic mycobacteria include the MAPKs p38 and ERK1/2 and the calmodulin/calmodulin kinase pathway (13, 14, 15, 16). Interestingly, the activation of these pathways, which is significantly elevated in macrophages infected with nonpathogenic Mycobacterium smegmatis, is dependent on intracellular Ca²⁺ (15). However, whether SPK activity is linked to the previously observed differences in MAPK activation seen in macrophages upon infection with M. smegmatis and Mycobacterium avium has not been addressed. Finally, it is not known whether SPK is important for the production of proinflammatory mediators by macrophages upon mycobacterial infection or whether there is a differential role for SPK upon infection with nonpathogenic and pathogenic mycobacteria.

We determined that SPK activity was required for ERK1/2, but not p38, MAPK activation following a M. avium or M. smegmatis infection of murine bone marrow-derived macrophages (BMM), and that the SPK functions along with phosphoinositide-specific phospholipase C (PI-PLC) and conventional protein kinase C (cPKC) to mediate ERK1/2 activation. PI-PLC, cPKC, and PI3K activity was elevated in BMM infected with nonpathogenic M. smegmatis compared with M. avium-infected cells. Moreover, the PI3K activity was also dependent on SPK/PI-PLC/PKC pathway. Finally, the increased production of proinflammatory mediators including TNF-, IL-6, RANTES, and G-CSF observed in M. smegmatis-infected BMM was dependent on the SPK/PI-PLC/PKC-mediated activation of ERK1/2 and PI3K. Our studies are the first to demonstrate a role for SPK in activating a macrophage proinflammatory response following a mycobacterial infection and a link between SPK and PI-PLC/PKC activation of ERK1/2 and PI3K in macrophages.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
BMM isolation and culture

BMM, used in all experiments, were isolated from 6- to 8-wk-old BALB/c mice as previously described (14, 17). Briefly, bone marrow was isolated and fibroblasts and mature macrophages were removed by selective adhesion. The isolated monocytes were cultured in DMEM (Invitrogen Life Technologies) containing 4.5 g/L D-glucose, 4.5 g/L L-glutamine, and 200 mg/ml CaCl₂, supplemented with 20 mM HEPES (Mediatech Cellgro), 10% FBS (Invitrogen Life Technologies), 100 U/ml penicillin and 100 µg/ml streptomycin (BioWhittaker), 1x L-glutamine (Mediatech Cellgro), and 20% L-Cell supernatant as a source of M-CSF. After 4 days in culture, BMM were supplied fresh medium and mature macrophages were harvested on day 7 and frozen at –140°C. Thawed macrophages were cultured on nontissue culture plates for 3–7 days, passaged, and allowed to recover for 3–6 days, and then replated at 3 x 10⁵ cells/35-mm tissue culture plates. The cells were allowed to adhere for 24 h before infection.

For all experiments, mycobacteria were added to macrophages on ice and incubated for 10 min, allowing mycobacteria to settle onto the cells, and then incubated at 37°C in 5% CO₂ for the specified times. Culture medium without antibiotics or L cell supernatant was used in place of complete medium during the infections. For the 24- and 48-h time points, the BMM were incubated for 4 h with the mycobacteria and vehicle controls or inhibitors, washed with PBS three times, then 2 ml of fresh medium was added with vehicle controls or inhibitors and incubated for a total of 24 or 48 h. For Ca2⁺-free medium, we used DMEM without CaCl₂ but containing the remaining ingredients. All tissue culture reagents were found negative for endotoxin contamination using either the E-Toxate assay (Sigma-Aldrich) or QCL-1000 Endotoxin test (Cambrex BioScience).

Treatment with pharmacological reagents

The inhibitors were purchased from Calbiochem, reconstituted in ethanol dihydrosphingosine (DHS) or in sterile DMSO (all other inhibitors), and used under the following conditions: DHS (25 µM), SPK inhibitor (SKI, 10 µM) (catalog no. 567731, 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole), Gö6976 (1 µM), Ro31-8425 (10 µM), and 2-aminoethoxydiphenyl borate (2-APB, 50 or 100 µM) were added 20 min before the infection, U73122 (2 µM), U73343 (2 µM), D-609 (10 µM), and LY294002 (50 µM) were added 30 min after the infection for 1 h infection or 2 h after for 24- or 48-h infection. DMSO or ethanol (DHS) was used in the same concentrations as the vehicle control. For all inhibitors, either a dose response was observed in relation to ERK1/2 phosphorylation and the concentrations used in subsequent studies were chosen based on the dose response or concentrations were chosen based on the previous studies published with macrophages (4, 18, 19, 20, 21). The 2 µM S1P (Calbiochem) dissolved in methanol was added to the macrophages 30 min before infection. For addressing the role of intracellular and extracellular calcium in BMM activation, cells were treated with BAPTA-AM (10 µM), (an intracellular calcium chelator), or EGTA (5 mM) to chelate extracellular calcium, 20 min before infection. All the reagents were tested and found not to have a significant effect on the uptake of the mycobacteria by the macrophages (data not shown).

Bacteria culture

To generate M. avium 724 stocks, the mycobacteria were passaged through a mouse to ensure virulence and a single colony was used to inoculate Middlebrooks 7H9 medium (Difco) supplemented with GOATS (glucose, oleic acid, albumin, Tween 20, and NaCl). Bacteria were grown for 10 days at 37°C with vigorous shaking, resuspended in Middlebrooks/GOATS with 15% glycerol, aliquoted, and stored at –80°C. Frozen stocks were quantitated by serial dilution onto Middlebrooks 7H10 agar/GOATS. M. smegmatis strain MC²155 from American Type Culture Collection was grown in Middlebrooks/GOATS at 37°C for 2–4 days. Frozen stocks were prepared as described for M. avium. All reagents used to grow mycobacteria were found negative for endotoxin contamination using the E-Toxate assay (Sigma-Aldrich) and the QCL-1000 Endotoxin test (Cambrex BioScience).

Complement opsonization

Appropriate concentrations of mycobacteria were suspended in macrophage culture medium containing 10% normal horse serum as a source of complement components and incubated for 2 h at 37°C (17). The same concentration of normal horse serum was added to uninfected controls for all experiments.

Mycobacteria infection

Infection assays performed on each stock of mycobacteria were evaluated by fluorescence microscopy to determine the infection ratio needed to obtain 80% of the macrophages infected. Briefly, BMM were plated on glass coverslips and infected with different doses of mycobacteria in triplicate. Infections were halted at either 1 or 4 h and fixed in 1:1 methanol:acetone, washed with PBS, and stained with TB Auramine M Stain kit (BD Biosciences) in the case of M. avium, and with acridine orange (Sigma-Aldrich) in the case of M. smegmatis. Slides were visualized using fluorescent microscopy and the level of infection was quantitated by counting the number of cells infected in at least four fields per replicate. No fewer than 100 cells per replicate were counted. Analogous infection assays were performed with or without inhibitors.

PKC activity assay

After infection with mycobacteria, the BMM were lysed with ice-cold lysis buffer as described below; cell lysates were removed and assayed for cPKC activity using the PKC activity assay kit (Upstate Biotechnology). The kinase reaction mixture contained 5 mM MOPS (pH 7.2), 5 mM -glyceraldehyde phosphate, 0.2 mM sodium orthovanadate, 0.2 mM DTT, 100 µM PKC substrate peptide, 100 µg/ml phosphatidylserine, 100 µg/ml 1,2-diacylglycerol (DAG) protein kinase A/calmodulin kinase inhibitor mix (provided with the kit), 15 mM MgCl₂, 100 µM ATP, and 5 µCi (3000 Ci/mmol) of [-³²P]ATP. Kinase reactions were initiated by the addition of freshly prepared cell lysates to the reaction mixture at 30°C. After 10 min, the reaction was terminated by spotting onto phosphocellulose paper (Whatman). The paper was washed three times with 0.75% phosphoric acid and finally with acetone. The [-³²P]ATP incorporation was measured using a scintillation counter (Beckman Coulter).

Measurement of inositol 1,4,5-trisphosphate (IP₃)

BMM were infected with mycobacteria in the presence of inhibitor or vehicle control. Three minutes after infection IP₃ formation was measured using a competitive radioactivity assay (Amersham Biosciences), according to manufacturer’s protocols. Briefly, after infection, cells were lysed in 0.2x volumes of 20% perchloric acid on ice for 20 min. After centrifugation at 2000 x g for 15 min at 4°C, supernatants were collected and neutralized (to pH 7.5) by titrating with ice-cold 1.5 M KOH containing 60 mM HEPES. Precipitated KClO₄ was removed by centrifugation at 2000 x g for 15 min at 4°C. Supernatants were collected and assayed for IP₃ formation with a competitive IP₃ binding assay using a fixed amount of IP₃ binding protein and radiolabeled IP₃ as described in the manufacturer’s protocol. The radioactivity was measured using a scintillation counter (Beckman Coulter).

Western blot analysis

At designated times, the treated BMM were removed from the incubator and placed on ice. The cells were washed three times with ice-cold PBS containing 1 mM pervanadate. The cells were then treated for 5–10 min with ice-cold lysis buffer (150 mM NaCl, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM pervanadate, 1 mM EDTA, 1% Igepal, 0.25% deoxycholic acid, 1 mM NaF, and 50 mM Tris-HCl (pH 7.4)). The cell lysates were removed from the plates and stored at –20°C. Equal amounts of protein, as defined using the Micro BCA Protein Assay (Pierce), were loaded onto 10% SDS-PAGE gels, electrophoresed and transferred onto polyvinylidene difluoride membrane (Millipore). The membranes were blocked in TBST (Tris-buffered saline with 0.05% Tween 20) supplemented with 5% powdered milk and then incubated with primary Abs against phospho-p38, phospho-ERK1/2, total ERK1/2, or phospho-Akt from Cell Signaling Technology. The blots were washed with TBST and incubated with a secondary Ab, either HRP-conjugated anti-rabbit or anti-mouse Ig (Pierce) in TBST plus 5% powdered milk. The bound Abs were detected using SuperSignal West Femto ECL reagents (Pierce).

ELISA

The levels of cytokines secreted by infected macrophages were measured using the commercially available ELISA reagent kits for TNF- (BD Pharmingen), IL-6, RANTES (eBioscience), and G-CSF (R&D Systems). Culture medium collected from the macrophages was analyzed for cytokines according to manufacturer’s instructions and the cytokine concentrations were determined against the standard curves. A cytokine profile analysis was performed by using the RayBio Mouse Cytokine Ab Array (RayBiotech) with culture supernatants from noninfected or infected macrophages (data not shown).

Statistical analysis

Statistical significance was determined with the paired two-tailed Student’s t test. Values at p < 0.05 level were considered significant and determined using InStat/Prism software.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
SPK is important for ERK1/2 activation and TNF- production in macrophages infected with mycobacteria

We showed previously that Ca²⁺ is important for the activation of macrophage signaling pathways upon mycobacterial infection (15). A study by Malik et al. (4) showed that infection with dead M. tuberculosis, but not live, leads to the activation of SPK in human macrophages. This activation of SPK was responsible for the induction of Ca²⁺ flux and phagosome maturation in macrophages infected with dead mycobacteria (4, 22). SPK catalyzes the formation of S1P from sphingosine and ATP, which then acts as a secondary messenger for the Ca²⁺ release (23). However, it is not known whether SPK is involved in other signaling pathways upon mycobacterial infection and whether there is a differential role for SPK in macrophages infected with nonpathogenic compared with pathogenic mycobacteria. To answer these questions, we looked at the role of SPK in macrophages infected with nonpathogenic M. smegmatis and pathogenic M. avium 724. Consistent with the previous reports, BMM infected with M. smegmatis show similar or slightly higher levels of MAPK activation compared with macrophages infected with M. avium at 30 min and 1 h postinfection (Fig. 1A). However, p38 and ERK1/2 activation is lost in macrophages infected with M. avium 724 between 1 and 2 h, whereas in M. smegmatis-infected macrophages there is a sustained activation of ERK1/2 and p38 (15). Next we treated BMM with SPK inhibitors DHS (24) and SKI (25) and looked at the activation of the MAPKs in mycobacteria-infected macrophages. DHS (25 µM) and SKI (10 µM) were added to the macrophages 20 min before infection and did not have any effect on the uptake of mycobacteria by the macrophages (data not shown). Treatment with DHS and SKI resulted in inhibition of ERK1/2 activation in macrophages infected with M. smegmatis or M. avium (Fig. 1, B and C). p38 Activation remained unaffected upon treatment with SPK inhibitors. Our published studies have shown that macrophages infected with fast-growing, nonpathogenic mycobacteria induce significantly higher TNF- compared with macrophages infected with pathogenic M. avium, and this response was dependent on MAPK activation (14). To investigate whether SPK is important for TNF- production, we pretreated macrophages with SKIs and measured TNF- secretion upon mycobacterial infection. As observed previously, M. smegmatis infection of BMM resulted in higher levels of TNF- production compared with M. avium-infected cells (Fig. 1D). This TNF- secretion was blocked in the presence of DHS and SKI indicating that SPK is required for the ERK1/2 activation and for TNF- production in M. smegmatis-infected and, to a lesser extent, M. avium-infected macrophages.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. SPK is required for ERK1/2 activation and TNF- production in macrophages infected with mycobacteria. A, ERK1/2 and p38 phosphorylation in macrophages infected with M. smegmatis and M. avium 724. BMM were infected with mycobacteria for 0.5, 1, 2, or 4 h and after infection, cells were lysed and the cell lysates were analyzed by Western blotting for activated ERK1/2 and p38 using phosphospecific Abs as described in Materials and Methods. BMM were pretreated with SPK inhibitors DHS (B) or SKI (C) or with ethanol (–) or DMSO (–) as vehicle controls, 20 min before infection with either M. smegmatis or M. avium 724. After a 1-h infection, BMM were lysed and the cell lysates were analyzed by Western blotting. Total ERK1/2 blots were run to show equal protein loading. D, Culture supernatants from 24 h infected and noninfected BMM were analyzed for TNF- by ELISA. Values are expressed as mean + SD. *, Significant (p < 0.01) to control. The results are representative of three separate experiments. RC, Resting, noninfected BMM; Smeg, M. smegmatis; Avium, M. avium 724.

Although SPK and S1P have been linked to ERK activation in a number of cell types, it has not been demonstrated in macrophages. In many systems, the ERK activation is through S1P-mediated activation of G protein-coupled receptors and subsequent activation of the Ras-ERK pathway (26, 27). However, a recent study by Blom et al. (28) showed that PI-PLC inhibitor U73122 blocked S1P-mediated Ca²⁺ release in HEK293 cells. Therefore, we were interested in investigating the potential involvement of PLC-PKC pathway in the SPK-mediated ERK1/2 activation in BMM infected with mycobacteria. We first tested whether PLC was involved in ERK activation by treating infected BMM with U73122 (PI-PLC inhibitor) or D609 (phosphatidylcholine-specific (PC)-PLC inhibitor). Inhibitors were added to macrophages 30 min postinfection and had no significant effect on phagocytosis (data not shown). As revealed in Fig. 2A, treatment with U73122 (2 µM), but not D609 (10 µM), results in inhibition of ERK1/2 activation in macrophages infected with mycobacteria. No inhibition of p38 activation was observed. To ensure the specificity of the inhibitor, we also treated macrophages with U73343 (2 µM), a structural analog of U73122, and found it to have no effect on ERK1/2 phosphorylation (data not shown). This result indicated that PI-PLC, but not PC-PLC, is important for ERK1/2 activation. Because the products of PI-PLC activation (i.e., IP₃ and DAG) function to stimulate cPKC, we examined the significance of these cPKC isoforms in MAPK activation. As shown in Fig. 2, B and C, ERK1/2 activation in macrophages following infection with mycobacteria was inhibited by Gö6976 (1 µM), a selective inhibitor of PKC- isozyme and PKC-1, and also by Ro31-8425 (10 µM), a selective inhibitor of PKC-, _I, _II, and isoforms. There was a dose-dependent inhibition of ERK1/2 activation upon treatment with inhibitors (Fig. 2C and data not shown). p38 Activation was not inhibited by Gö6976 or Ro31-8425 treatment in either M. smegmatis- or M. avium-infected macrophages.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. PI-PLC and cPKC function upstream of ERK1/2 activation in macrophages infected with mycobacteria. A, BMM were treated with PI-PLC inhibitor U73122 and PC-PLC inhibitor D609 or with DMSO (–) as the vehicle control, 30 min postinfection with M. smegmatis or M. avium. BMM were infected for a total of 1 h. To determine the involvement of PKC in MAPK activation, BMM were treated with PKC inhibitors Gö6976 (B) or Ro31-8425 (C) or with DMSO (-) as the vehicle control, 20 min before a 1-h infection with M. smegmatis or M. avium 724. Macrophages were lysed and the cell lysates were analyzed by Western blot for activated ERK1/2 and p38 using phosphospecific Abs as described in Materials and Methods. Total ERK1/2 blots were run to show equal protein loading. The results are representative of three separate experiments. RC, Resting, noninfected BMM.

PI-PLC and SPK mediate PKC activation leads to ERK1/2 phosphorylation in mycobacteria-infected BMM

We hypothesized that SPK functions through PI-PLC and PKC to mediate ERK1/2 activation in BMM following a mycobacterial infection. To test this possibility, we measured the kinase activity of cPKC in BMM infected with M. smegmatis and M. avium 724 and examined the effect of SPK and PI-PLC inhibitors on the kinase activity. We found that PKC activity is significantly higher in macrophages infected with M. smegmatis compared with M. avium-infected or noninfected macrophages at 1 h postinfection (Fig. 3A). BMM infected with M. avium showed a slight increase in PKC activity above resting cells (RC, 111 ± 3%), but it was not statistically significant (p > 0.05). PKC activation induced upon mycobacterial infection was blocked when macrophages were pretreated with Gö6976 (data not shown) or Ro31-8425 (Fig. 3A), demonstrating the specificity of the kinase assay for the cPKC. We saw a similar trend in PKC activation after 30 min and 2 h of infection (data not shown). As expected, because PKC is activated by the products of PI-PLC (i.e., DAG- and IP₃-mediated Ca²⁺ release), we found the PKC activation induced upon M. smegmatis infection to be inhibited by treating the BMM with the PI-PLC inhibitor U73122 (Fig. 3B). The minimal PKC activation above RC levels observed in M. avium-infected macrophages was also inhibited in the presence of U73122 (from 110 ± 3% to 100 ± 8%); however, again the levels were not statistically different. To test whether SPK is present upstream of the PKC pathway, we tested the effect of DHS on PKC activation. Similar to what was seen with PI-PLC inhibitors, PKC activation induced upon M. smegmatis infection was inhibited in the presence of DHS (Fig. 3C). Following an M. avium infection, PKC activation was diminished to RC levels upon treatment with DHS (from 110 + 5% to 98 + 0.3%) (Fig. 3C). These experiments, however, did not determine whether PI-PLC lies upstream or downstream of SPK activation. To define where PI-PLC resides in the ERK1/2 activation pathway, we infected BMM in the presence of the SKI and measured IP₃ levels. As shown in Fig. 3D, the addition of the SKI to M. smegmatis-infected BMM lead to a significant decrease in IP₃ formation, suggesting that PI-PLC activation is dependent on SPK. M. avium infection did not lead to a significant increase in IP₃ levels relative to noninfected macrophages (Fig. 3D).

View larger version (22K): [in this window] [in a new window]   FIGURE 3. cPKC activity is elevated in M. smegmatis-infected BMM and is dependent on SPK and PI-PLC. A, BMM infected for 1 h or left noninfected were lysed, and the cell lysates were assayed for PKC activity as described in Materials and Methods. Before the PKC activity assay, one well of M. smegmatis-infected macrophages was treated with Ro31-8425 (A) as described in Fig. 2. BMM were infected with M. smegmatis or M. avium with (+) or without (–) U73122 (B), DHS (C), or SKI (D) as described in Figs. 1 and 2 and the cell lysates were assayed for PKC activity (C) or IP3 levels (D). IP3 concentrations were measured as described in Materials and Methods. Values are expressed as mean + SD. *, Significant (p < 0.05) to RC, **, Significant (p < 0.05) to Smeg (–). Data are representative of three separate experiments. RC, Resting, noninfected BMM; Ro, Ro31-8425; Smeg, M. smegmatis; Avium, M. avium 724.

SPK mediates BMM activation through release of intracellular calcium

S1P generated through SPK activity might be functioning extracellularly to stimulate IP₃ formation by activating G protein-coupled receptors, as previously observed (7), or by working directly to stimulate PI-PLC activity. To address these possibilities, we performed the infection experiments in the presence of SKI or DHS with or without extracellular S1P. We observed an inhibition of ERK1/2 activation and TNF- production in the presence of SPK inhibitors, and this effect was not reversed by incubation with S1P, suggesting that the effect of SPK on BMM activation is not dependent on extracellular S1P (Fig. 4A and data not shown). The lack of an effect by extracellular S1P and a known role for IP₃ in the release of Ca²⁺ from the endoplasmic reticulum suggest that it is the intracellular pool of Ca²⁺ that is released initially upon mycobacterial infection and is required for MAPK activation. However, to test this suggestion more directly, infection experiments were performed in Ca²⁺-free medium or in the presence of EGTA to eliminate the source of extracellular Ca²⁺ or incubated with BAPTA-AM to chelate intracellular Ca²⁺. As expected, removal of Ca²⁺ from the extracellular medium did not effect TNF- secretion (Fig. 4B), MAPK activation (Fig. 4C), or IL-6 release (data not shown) by BMM following infection. In contrast, chelation of intracellular Ca²⁺ with BAPTA-AM resulted in the loss of ERK activation and cytokine production as we have observed previously (15) (Fig. 4, B and C). Further evidence indicating that IP₃-mediated release of intracellular Ca²⁺ is required for BMM activation following a mycobacterial infection stems from studies using 2-APB, an inhibitor of IP₃-induced Ca²⁺ release. The 2-APB inhibitor significantly blocked, in a dose-dependent manner, TNF- production in both M. smegmatis- and M. avium-infected BMM (Fig. 4D).

View larger version (34K): [in this window] [in a new window]   FIGURE 4. SPK-mediated activation of BMM is not dependent on extracellular S1P but is dependent on IP3-mediated release of intracellular Ca2+. A, BMM were infected with M. smegmatis or M. avium in the presence or absence of SPK inhibitors DHS or SKI as described in Fig. 1. BMM were also incubated with (+) or without (–) S1P and with or without SKI, and TNF- levels in the culture supernatants were measured 24 h postinfection by ELISA. B and C, BMM were infected with M. smegmatis or M. avium in the presence of BAPTA-AM, Ca2+-free culture medium, or EGTA and assayed for TNF- production and MAPK activation. D, BMM were infected with mycobacteria in the presence of 2-APB (50 or 100 µM), an inhibitor of IP3-induced Ca2+ release. After infection, culture supernatants were removed and analyzed for TNF- production. Values are expressed as mean + SD. *, Significant (p < 0.05) to control; ns, not significant (p > 0.05) to control. Data are representative of three separate experiments. RC, Resting, noninfected BMM; Smeg, M. smegmatis; Avium, M. avium 724.

PI3K has been shown to stimulate ERK1/2 activation in macrophages (29, 30). To determine whether PI3K is activated upon a mycobacterial infection, we measured Akt/protein kinase B phosphorylation at serine 473. This residue is a well-known substrate for PI3K phosphorylation. We observed a more pronounced Akt phosphorylation in macrophages infected with M. smegmatis compared with M. avium-infected or noninfected cells (Fig. 5A). This activation was inhibited when macrophages were treated with PI3K inhibitor LY294002 (50 µM). However, there was no effect on ERK1/2 or p38 activation upon treatment with LY294002, indicating that PI3K is not present upstream of MAPK pathways. The LY294002 was added to the macrophages 30 min after infection due to its known effect on phagocytosis. Under these conditions, the LY294002 did not significantly affect phagocytosis. We also measured Akt phosphorylation in the presence of MAPK inhibitors. As expected, PI3K activation (measured through Akt phosphorylation) was not affected in macrophages treated with MEK1/2 inhibitor PD98059 (Fig. 5B) or p38 MAPK inhibitor III (data not shown), suggesting that the PI3K is parallel to the MAPK pathway. We also tested whether the SPK, PI-PLC, and PKC pathways were upstream of PI3K activation. For these experiments, we treated mycobacteria-infected BMM with the various inhibitors: DHS for SPK, U73122 for PI-PLC, and Gö6976 for PKC, and looked at phosphorylation of Akt. As shown in Fig. 5, C and D, PI3K activation was inhibited in the presence of all inhibitors placing SPK/PI-PLC/PKC upstream of PI3K.

View larger version (44K): [in this window] [in a new window]   FIGURE 5. PI3K activity in Mycobacterium-infected BMM is dependent on the SPK/PI-PLC/PKC pathway but not on MEK1/2 activity. A, BMM were treated with PI3K inhibitor LY294002 30 min postinfection and the infection continued for a total of 1 h. Cell lysates were analyzed by Western blot for Akt phosphorylation, which was used as a measure of PI3K activity. In separate experiments, BMM were also treated with MEK1/2 inhibitor PD98059 (B), PKC inhibitor Gö6976 (C), SPK inhibitor DHS (C), or PI-PLC inhibitor U73122 (D), or the vehicle control (–) as described in Figs. 1 and 2. After mycobacterial infections, cells were lysed and the cells lysates analyzed for activated Akt, ERK, or p38 using phosphospecific Abs as described in Materials and Methods. Total ERK1/2 blots were run to show equal protein loading. These results are representative of three separate experiments. RC, Resting, noninfected BMM.

PI-PLC, PKC, and PI3K are required for TNF- production in macrophages infected with M. smegmatis and M. avium

As shown in Fig. 1C, SPK is required for TNF- production in macrophages infected with mycobacteria. Because we found a role for PI-PLC and PKC in ERK1/2 activation and a link between SPK and the PI-PLC/PKC pathway, we examined the significance of these signaling molecules in TNF- production following infection with M. smegmatis and M. avium. We also tested whether PI3K was required for TNF- production because we observed its differential activation in macrophages infected with M. smegmatis compared with M. avium. As shown in Fig. 6A, the high TNF- production induced in M. smegmatis-infected macrophages was inhibited in the presence of U73122, Gö6976, Ro31-8425, and LY294002. The low TNF- production in M. avium-infected macrophages was also inhibited after treatment with the various inhibitors; however, there was only a 20–30% drop in TNF- production with LY294002, which was not statistically significant (p > 0.05). The results underscore the importance of these signaling molecules in promoting TNF- production by BMM upon infection with mycobacteria and how the differential activation of these signaling molecules could be responsible for the differences in TNF- production observed between BMM infected with M. smegmatis and with M. avium.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. A role for SPK, PKC, PI3K, and PI-PLC in the production of TNF- (A), G-CSF (B), IL-6 (C), and RANTES (D) production by BMM upon infection with mycobacteria. BMM were pretreated with different inhibitors or the vehicle control as described in Figs. 1, 2, and 4, and infected with M. smegmatis and M. avium 724 for 4 h. The cells were washed with PBS and the infection continued for a total of 24 h (for TNF-) or 48 h (for G-CSF, IL-6, and RANTES). After infection, culture supernatants were removed and analyzed by ELISA. Values are expressed as mean + SD. ns, Not significant (p > 0.05) to control M. avium. Data are representative of three separate experiments. RC, Resting, noninfected BMM.

To test whether activation of these signaling molecules is required for other macrophage responses to mycobacterial infection, we looked at the production of additional inflammatory mediators following treatment with the different inhibitors. However, we first investigated, using a RayBio Mouse Cytokine Ab Array, whether production of other cytokines and chemokines differed between macrophages infected with M. smegmatis or with M. avium. We found that, in addition to TNF-, M. smegmatis-infected macrophages showed increased levels of IL-6, G-CSF, and RANTES compared with M. avium 724-infected cells (data not shown). A difference in macrophage production was also observed by ELISA (Fig. 6, B–D). Furthermore, as shown in Fig. 6, B–D, levels of all three of these inflammatory mediators were significantly decreased in M. smegmatis-infected BMM treated with the SPK, PI-PLC, and PKC inhibitors, although the PKC inhibitors Gö6976 and Ro31-8425 blocked production of G-CSF and RANTES only 60–70%. However, both SPK inhibitors DHS and SKI completely or almost completely blocked production of all three mediators. The PI3K inhibitor LY294002 completely blocked G-CSF and IL-6 but inhibited RANTES production only 60–70% (Fig. 5, B–D). In M. avium-infected BMM, there was a little production of G-CSF and IL-6, which were at undetectable levels in the presence of inhibitors. However, there was a significant amount of RANTES secreted in M. avium-infected BMM, and it was blocked completely by DHS and SKI, whereas LY294002, Gö6976, and Ro31-8425 resulted in 40–60% drop (Fig. 6D).

	Discussion

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Pathogenic mycobacteria successfully reside inside host macrophages by inhibiting several host cell processes. A number of macrophage cellular functions have been shown to be inhibited by pathogenic mycobacteria, including the fusion of phagosome with lysosome, Ag presentation, apoptosis, and the stimulation of antimicrobial responses due to activation of pathways involving MAPKs, IFN-, and Ca²⁺ signaling. This modulation of host cell signaling is critical for the persistence of mycobacteria within the host and for the suppression of a generalized inflammatory response (reviewed in Refs.12 and 31). Recently, a number of studies have shown that pathogenic mycobacteria inhibit macrophages function by blocking Ca²⁺-dependent signaling (3, 4, 32). In primary human macrophages, live M. tuberculosis fails to promote SPK activity and inhibits SPK activity induced upon infection with heat-killed M. tuberculosis. This lack of SPK stimulation in macrophages infected with live M. tuberculosis results in a reduced cytosolic Ca²⁺ concentration. This Ca²⁺ induction is necessary for phagosome maturation (4). A recent study has shown that engagement of the mannose receptor by mannose-lipoarabinomannan, which is normally expressed by M. tuberculosis, is important in restricting phagosome-lysosome fusion (33). Whether there is a link between engagement of the mannose receptor by M. tuberculosis and the bacilli’s ability to inhibit SPK activity warrants further study.

SPK phosphorylates a host lipid, sphingosine, to form S1P, which is a ligand for specific G protein-coupled receptors and also regulates intracellular Ca²⁺ homeostasis by releasing Ca²⁺ from the cytoplasmic organelles (23, 34). SPK is activated by various growth factors and cytokines, including FCS, platelet-derived growth factor (24), basic fibroblast growth factor (35), TNF- (36), IL-1 (37), and vascular endothelial growth factor (26).

Our previous studies indicated that Ca²⁺ induction was also important for MAPK activation mediated through activation of calmodulin and calmodulin kinase and that this activation was elevated in macrophages infected with nonpathogenic mycobacteria relative to cells infected with M. avium (15). However, the role played by SPK in the Ca²⁺ induction and MAPK activation was not addressed in this study. Moreover, how SPK induces a Ca²⁺ flux also was not defined in previous work. We hypothesized that SPK could be involved in MAPK activation and might be responsible for the sustained MAPK activation and increased TNF- production seen in macrophage infected with nonpathogenic mycobacteria. As predicted, the SPK inhibitors DHS and SKI blocked the nonpathogenic M. smegmatis and virulent M. avium 724 induced activation of ERK1/2 by infected macrophages. A similar role for SPK has been proposed in LPS-mediated ERK1/2 activation in RAW 264.7 cells (38). This also correlates well with our previous data in which we found a role for Ca²⁺ in ERK1/2 activation. SPK has previously been implicated in generation of inflammatory mediators in macrophages (39). Blocking SPK with DHS and SKI also resulted in a significant drop in TNF- production upon infection with both M. smegmatis and M. avium 724. Chelating intracellular Ca²⁺ or blocking Ca²⁺ release from the endoplasmic reticulum had a similar effect on TNF- production. This effect was more pronounced in M. smegmatis-infected BMM, suggesting that SPK is required for the enhanced TNF- production observed in macrophages infected with nonpathogenic mycobacteria. Together, these suggest that a Ca²⁺ flux following a mycobacterial infection is required for optimum macrophage activation and that SPK mediates this calcium flux.

SPK promotes ERK1/2 activation in variety of cell types through various mechanisms (36, 40). In endothelial cells and tumor cells, it was shown that SPK mediates vascular endothelial growth factor-induced activation of Ras, Raf, and ERK1/2 by down-regulating Ras-GTPase-activating protein activity (26, 41). SPK can mediate its effect as a second messenger both dependent and independent of PLC pathway (reviewed in Ref.42). S1P, the product of SPK activation, can mobilize the Ca²⁺ channels directly or can activate the PLC-dependent pathways by activating G protein-coupled receptors (7). However, the mechanism by which SPK stimulates Ca²⁺ release in macrophages under various stimuli has not been defined. Therefore, we tested whether the PLC-PKC signaling pathway was involved in the MAPK activation upon mycobacterial infection and whether the SPK-mediated ERK1/2 activation involved the PLC-PKC pathway. Both PC-PLC and PI-PLC can stimulate MAPK activation in macrophages depending on the stimulus. In human macrophages, PC-PLC, but not PI-PLC, is required for LPS-induced MAPK activation (43). Interestingly, we found that PI-PLC, but not PC-PLC, is required for ERK1/2 activation in macrophages upon mycobacterial infection. The products of PI-PLC activation (i.e., DAG and IP₃) act as secondary messengers for the activation of cPKC isoforms. Therefore, we investigated activation of cPKC upon infection with mycobacteria.

PKC was originally described as a Ca²⁺- and phospholipid-dependent protein kinase activated by DAG and other lipids. The PKC family is divided into three groups: 1) cPKCs comprise the , _I, _II, and isoforms and are dependent on Ca²⁺ and DAG; 2) novel PKCs, including the PKC-, , , and isoforms, are Ca²⁺ independent and regulated by DAG and phosphatidylserine; and 3) atypical PKCs, including PKC- and PKC- isoforms, are Ca²⁺ and DAG independent (44, 45). PKCs have been shown to be activated during phagocytosis and play an important role in phagosome maturation and immune signaling (46, 47, 48, 49). Some PKC isoforms are implicated in FcR-mediated phagocytosis but cPKCs have been shown not to be required for phagocytosis (50). As expected, we found a similar effect of cPKC inhibitors on ERK1/2 activation as seen with the PI-PLC and SPK inhibitors. Our data showing a lack of ERK1/2 activation in BMM treated with cPKC inhibitors Gö6976 and Ro31-8425 correlated well with other studies in which cPKC was required for LPS induced ERK1/2 activation in macrophages (19, 51). However, p38 activation was not affected by cPKC inhibition, perhaps due to the regulation of p38 by other isoforms of PKC, as shown previously with LPS-induced JNK but not p38 activation, being dependent on PKC- (52). Also as predicated, we found PKC activity to be elevated in macrophages infected with M. smegmatis compared with uninfected or M. avium-infected BMM. This cPKC activity was blocked upon pretreating macrophages with Gö6976 or Ro31-8425 confirming the specificity of the assay for cPKCs. The high background of cPKC activity in noninfected BMM likely results from the kinases’ role in maintaining cellular adhesion. It also suggests that the increase in cPKC activity is required for the prolonged ERK1/2 activation seen in M. smegmatis-infected macrophages (i.e., 2 and 4 h postinfection). To our knowledge this is the first report showing differential activation of cPKC upon infection with pathogenic and nonpathogenic mycobacteria. A recent study indicated a similar activation of PKC- by noncapsulated mutant Streptococcus suis but not by pathogenic encapsulated bacteria (53). cPKC have also been shown to be activated in macrophages upon stimulation with cord factor from M. tuberculosis (54). In our studies, we found cPKC activity to be inhibited when BMM were pretreated with the PI-PLC inhibitor U73122. It was also lowered to the RC levels upon pretreatment of macrophages with the SPK inhibitor DHS. Moreover, IP₃ formation, which is induced significantly in M. smegmatis-infected macrophages, is inhibited in the presence of SKI. These results demonstrate that SPK mediates cPKC activation through activation of PI-PLC. The exact mechanism of this activation remains unclear; however, it does not appear to involve release of S1P and activation of G protein-coupled receptors because the addition of extracellular S1P did not overcome the DHS- or SKI-mediated inhibition of macrophage activation.

PKC and MAPK activation have also been closely linked to PI3K stimulation. PI3K is a conserved family of signaling molecules that are involved in regulating cellular proliferation and survival. Recent data suggest that the PI3K-Akt pathway is activated in macrophages upon mycobacterial infection or treatment with mycobacterial glycolipids (55, 56, 57). We show that PI3K activity, as defined by Akt phosphorylation, was activated at higher levels upon infection with M. smegmatis compared with M. avium-infected macrophages. PI3K activity was dependent on the SPK, PI-PLC, and PKC but not on ERK1/2 or p38. This finding suggests that the SPK-PLC-PKC pathway mediates ERK1/2 and PI3K activation following infection with mycobacteria (Fig. 7).

View larger version (26K): [in this window] [in a new window]   FIGURE 7. Conceptual model for the signal transduction cascade initiated in primary BALB/c BMM following infection by M. smegmatis and M. avium 724. In BMM infected with mycobacteria, SPK, PI-PLC, and cPKC are required for ERK1/2 activation. There is a stronger activation of cPKCs and PI-PLC in M. smegmatis-infected BMM compared with M. avium 724-infected cells. Activation of PI-PLC and cPKC following the mycobacterial infections is dependent on SPK, and is required for ERK1/2 phosphorylation. Also, PI3K activity is up-regulated in M. smegmatis- compared with M. avium-infected BMM as determined by Akt phosphorylation. PI3K activation is dependent on the SPK/PI-PLC/PKC pathway but does not require ERK1/2 activation. SPK, PI-PLC, PKC, PI3K, and ERK1/2 activation are required for the production of TNF-, IL-6, G-CSF, and RANTES upon mycobacterial infection. However, there appears to be an elevated activation of these pathways upon M. smegmatis infection resulting in elevated production of proinflammatory mediators.

Previously, we showed that M. smegmatis infection leads to increased activation of the calmodulin/calmodulin kinase and cAMP-protein kinase A pathways, which are also required for ERK1/2 activation and up-regulation of TNF- production in BMM (15). We extend these findings in the present study by investigating the role of SPK, PLC, PKC, and PI3K in MAPK activation and production of inflammatory mediators. We show that nonpathogenic mycobacteria, and, to a more limited extent, M. avium use the SPK/PLC/PKC pathway to enhance the production of TNF-, G-CSF, IL-6, and RANTES. These are the first studies in macrophages to connect SPK to PI-PLC, PKC, ERK1/2, and PI3K activation following a mycobacterial infection. Our studies are also the first to define an important role for SPK in mediating macrophage activation to a live mycobacterial infection.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported through Grants AI056979 and AI052439 from the National Institute of Allergy and Infectious Diseases.

² Address correspondence and reprint requests to Dr. Jeffrey S. Schorey, Department of Biology, University of Notre Dame, 130 Galvin Life Science Center, Notre Dame, IN 46556. E-mail address: Schorey.1{at}nd.edu

³ Abbreviations used in this paper: SPK, sphingosine kinase; SKI, sphingosine kinase inhibitor; S1P, sphingosine-1-phosphate; BMM, bone marrow-derived macrophage; DHS, dihydrosphingosine; RC, resting cell; PLC, phospholipase C; PI-PLC, phosphoinositide-specific PLC; PC-PLC, phosphatidylcholine-specific PLC; PKC, protein kinase C; cPKC, conventional PKC; IP₃, inositol 1,4,5-trisphosphate; DAG, 1,2-diacylglycerol; 2-APB, 2-aminoethoxydiphenyl borate.

Received for publication September 8, 2005. Accepted for publication February 8, 2006.

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