The Journal of Immunology

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

Mahesh Yadav, Lindsay Clark, and Jeffrey S. Schorey2

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. The Journal of Immu- nology, 2006, 176: 5494–5503.

s intramacrophage pathogens, mycobacteria have SPK (4). SPK is a key enzyme catalyzing the formation of sphin- evolved multiple mechanisms to promote their entry and gosine-1-phosphate (S1P), a lipid messenger that is implicated in A survival in host cells. One well-characterized modifica- the regulation of a wide variety of important cellular events tion of host macrophages by mycobacteria is the inhibition of through both intracellular and extracellular mechanisms, which in- phagosome/lysosome fusion and avoidance of the degradative en- clude Ca2ϩ mobilization, activation of MAPK, and vesicular traf- vironment of a phagolysosome. In recent years, this ability of ficking (5–9). pathogenic mycobacteria to inhibit phagosome maturation has Infection by mycobacteria leads to a signaling response by the been under extensive study (for reviews see Refs. 1 and 2). Inter- host macrophage and subsequent production of proinflammatory estingly, one of the initial cellular events disrupted by Mycobac- mediators. However, our studies as well as others indicate that terium tuberculosis is the transient elevation of intracellular Ca2ϩ. macrophages infected with pathogenic mycobacteria produce sig- This calcium flux is required for the subsequent phagosome/lyso- nificantly less TNF-␣ and other proinflammatory molecules com- some fusion and induction of a Ca2ϩ flux upon an M. tuberculosis pared with cells infected with nonpathogenic mycobacteria (10, infection leads to phagosome maturation (3). This calcium rise, 11). A modulation of host cell signaling responses is critical for the which occurs upon infection with dead M. tuberculosis or live suppression of a generalized inflammatory response and the per- Staphylococcus aureus, is dependent on sphingosine kinase sistence of mycobacteria within the host (12). Macrophage signal- (SPK)3 activation. In contrast, live M. tuberculosis fails to activate ing pathways that are differentially activated by infection with pathogenic compared with nonpathogenic mycobacteria include the MAPKs p38 and ERK1/2 and the calmodulin/calmodulin ki- Department of Biological Sciences, Center for Tropical Disease Research and Train- ing, University of Notre Dame, Notre Dame, IN 46556 nase pathway (13–16). Interestingly, the activation of these path- Received for publication September 8, 2005. Accepted for publication February ways, which is significantly elevated in macrophages infected with 8, 2006. nonpathogenic Mycobacterium smegmatis, is dependent on intra- 2ϩ The costs of publication of this article were defrayed in part by the payment of page cellular Ca (15). However, whether SPK activity is linked to the charges. This article must therefore be hereby marked advertisement in accordance previously observed differences in MAPK activation seen in mac- with 18 U.S.C. Section 1734 solely to indicate this fact. rophages upon infection with M. smegmatis and Mycobacterium 1 This work was supported through Grants AI056979 and AI052439 from the Na- avium has not been addressed. Finally, it is not known whether tional Institute of Allergy and Infectious Diseases. SPK is important for the production of proinflammatory mediators 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, by macrophages upon mycobacterial infection or whether there is IN 46556. E-mail address: [email protected] 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, phos- kinase C; cPKC, conventional PKC; IP3, inositol 1,4,5-trisphosphate; DAG, 1,2-di- phoinositide-specific PLC; PC-PLC, phosphatidylcholine-specific PLC; PKC, protein acylglycerol; 2-APB, 2-aminoethoxydiphenyl borate.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 5495

a differential role for SPK upon infection with nonpathogenic and Bacteria culture pathogenic mycobacteria. To generate M. avium 724 stocks, the mycobacteria were passaged through We determined that SPK activity was required for ERK1/2, but a mouse to ensure virulence and a single colony was used to inoculate not p38, MAPK activation following a M. avium or M. smegmatis Middlebrooks 7H9 medium (Difco) supplemented with GOATS (glucose, infection of murine bone marrow-derived macrophages (BMM␾), oleic acid, albumin, Tween 20, and NaCl). Bacteria were grown for 10 days and that the SPK functions along with phosphoinositide-specific at 37°C with vigorous shaking, resuspended in Middlebrooks/GOATS with 15% glycerol, aliquoted, and stored at Ϫ80°C. Frozen stocks were quan- phospholipase C (PI-PLC) and conventional protein kinase C titated by serial dilution onto Middlebrooks 7H10 agar/GOATS. M. smeg- (cPKC) to mediate ERK1/2 activation. PI-PLC, cPKC, and PI3K matis strain MC2155 from American Type Culture Collection was grown activity was elevated in BMM␾ infected with nonpathogenic M. in Middlebrooks/GOATS at 37°C for 2–4 days. Frozen stocks were pre- smegmatis compared with M. avium-infected cells. Moreover, the pared as described for M. avium. All reagents used to grow mycobacteria were found negative for endotoxin contamination using the E-Toxate assay PI3K activity was also dependent on SPK/PI-PLC/PKC pathway. (Sigma-Aldrich) and the QCL-1000 Endotoxin test (Cambrex BioScience). Finally, the increased production of proinflammatory mediators including TNF-␣, IL-6, RANTES, and G-CSF observed in M. Complement opsonization smegmatis-infected BMM␾ was dependent on the SPK/PI-PLC/ Appropriate concentrations of mycobacteria were suspended in macro- PKC-mediated activation of ERK1/2 and PI3K. Our studies are the phage culture medium containing 10% normal horse serum as a source of first to demonstrate a role for SPK in activating a macrophage complement components and incubated for2hat37°C (17). The same proinflammatory response following a mycobacterial infection and concentration of normal horse serum was added to uninfected controls for all experiments. a link between SPK and PI-PLC/PKC activation of ERK1/2 and PI3K in macrophages. Mycobacteria infection Infection assays performed on each stock of mycobacteria were evaluated by fluorescence microscopy to determine the infection ratio needed to ob- Materials and Methods ϳ ␾ ␾ tain 80% of the macrophages infected. Briefly, BMM were plated on BMM isolation and culture glass coverslips and infected with different doses of mycobacteria in trip- BMM␾, used in all experiments, were isolated from 6- to 8-wk-old licate. Infections were halted at either 1 or 4 h and fixed in 1:1 methanol: BALB/c mice as previously described (14, 17). Briefly, bone marrow was acetone, washed with PBS, and stained with TB Auramine M Stain kit (BD isolated and fibroblasts and mature macrophages were removed by selec- Biosciences) in the case of M. avium, and with acridine orange (Sigma- tive adhesion. The isolated monocytes were cultured in DMEM (Invitrogen Aldrich) in the case of M. smegmatis. Slides were visualized using fluo- Life Technologies) containing 4.5 g/L D-glucose, 4.5 g/L L-glutamine, and rescent microscopy and the level of infection was quantitated by counting the number of cells infected in at least four fields per replicate. No fewer 200 mg/ml CaCl2, supplemented with 20 mM HEPES (Mediatech Cellgro), 10% FBS (Invitrogen Life Technologies), 100 U/ml penicillin and 100 than 100 cells per replicate were counted. Analogous infection assays were ␮g/ml streptomycin (BioWhittaker), 1ϫ L-glutamine (Mediatech Cellgro), performed with or without inhibitors. and 20% L-Cell supernatant as a source of M-CSF. After 4 days in culture, PKC activity assay BMM␾ were supplied fresh medium and mature macrophages were har- vested on day 7 and frozen at Ϫ140°C. Thawed macrophages were cultured After infection with mycobacteria, the BMM␾ were lysed with ice-cold on nontissue culture plates for 3–7 days, passaged, and allowed to recover lysis buffer as described below; cell lysates were removed and assayed for for 3–6 days, and then replated at ϳ3 ϫ 105 cells/35-mm tissue culture cPKC activity using the PKC activity assay kit (Upstate Biotechnology). plates. The cells were allowed to adhere for 24 h before infection. The kinase reaction mixture contained 5 mM MOPS (pH 7.2), 5 mM For all experiments, mycobacteria were added to macrophages on ice ␤-glyceraldehyde phosphate, 0.2 mM sodium orthovanadate, 0.2 mM DTT, and incubated for 10 min, allowing mycobacteria to settle onto the cells, 100 ␮M PKC substrate peptide, 100 ␮g/ml phosphatidylserine, 100 ␮g/ml and then incubated at 37°C in 5% CO2 for the specified times. Culture 1,2-diacylglycerol (DAG) protein kinase A/calmodulin kinase inhibitor ␮ ␮ medium without antibiotics or L cell supernatant was used in place of mix (provided with the kit), 15 mM MgCl2, 100 M ATP, and 5 Ci (3000 complete medium during the infections. For the 24- and 48-h time points, Ci/mmol) of [␥-32P]ATP. Kinase reactions were initiated by the addition of the BMM␾ were incubated for 4 h with the mycobacteria and vehicle freshly prepared cell lysates to the reaction mixture at 30°C. After 10 min, controls or inhibitors, washed with PBS three times, then 2 ml of fresh the reaction was terminated by spotting onto phosphocellulose paper medium was added with vehicle controls or inhibitors and incubated for a (Whatman). The paper was washed three times with 0.75% phosphoric acid ϩ 32 total of 24 or 48 h. For Ca2 -free medium, we used DMEM without CaCl2 and finally with acetone. The [␥- P]ATP incorporation was measured us- but containing the remaining ingredients. All tissue culture reagents were ing a scintillation counter (Beckman Coulter). found negative for endotoxin contamination using either the E-Toxate as- say (Sigma-Aldrich) or QCL-1000 Endotoxin test (Cambrex BioScience). Measurement of inositol 1,4,5-trisphosphate (IP3) BMM␾ were infected with mycobacteria in the presence of inhibitor or Treatment with pharmacological reagents vehicle control. Three minutes after infection IP3 formation was measured using a competitive radioactivity assay (Amersham Biosciences), accord- The inhibitors were purchased from Calbiochem, reconstituted in ethanol ing to manufacturer’s protocols. Briefly, after infection, cells were lysed in dihydrosphingosine (DHS) or in sterile DMSO (all other inhibitors), and 0.2ϫ volumes of 20% perchloric acid on ice for 20 min. After centrifu- used under the following conditions: DHS (25 ␮M), SPK inhibitor (SKI, gation at 2000 ϫ g for 15 min at 4°C, supernatants were collected and 10 ␮M) (catalog no. 567731, 2-( p-hydroxyanilino)-4-( p-chlorophenyl) neutralized (to pH 7.5) by titrating with ice-cold 1.5 M KOH containing 60 thiazole), Go¨6976 (1 ␮M), Ro31-8425 (10 ␮M), and 2-aminoethoxydiphe- mM HEPES. Precipitated KClO was removed by centrifugation at 2000 ϫ nyl borate (2-APB, 50 or 100 ␮M) were added 20 min before the infection, 4 g for 15 min at 4°C. Supernatants were collected and assayed for IP U73122 (2 ␮M), U73343 (2 ␮M), D-609 (10 ␮M), and LY294002 (50 3 formation with a competitive IP binding assay using a fixed amount of IP ␮M) were added 30 min after the infection for 1 h infection or 2 h after for 3 3 binding protein and radiolabeled IP as described in the manufacturer’s 24- or 48-h infection. DMSO or ethanol (DHS) was used in the same 3 protocol. The radioactivity was measured using a scintillation counter concentrations as the vehicle control. For all inhibitors, either a dose re- (Beckman Coulter). sponse was observed in relation to ERK1/2 phosphorylation and the con- centrations used in subsequent studies were chosen based on the dose re- Western blot analysis sponse or concentrations were chosen based on the previous studies published with macrophages (4, 18–21). The 2 ␮M S1P (Calbiochem) At designated times, the treated BMM␾ were removed from the incubator dissolved in methanol was added to the macrophages 30 min before in- and placed on ice. The cells were washed three times with ice-cold PBS fection. For addressing the role of intracellular and extracellular calcium in containing 1 mM pervanadate. The cells were then treated for 5–10 min BMM␾ activation, cells were treated with BAPTA-AM (10 ␮M), (an in- with ice-cold lysis buffer (150 mM NaCl, 1 mM PMSF, 1 ␮g/ml aprotinin, tracellular calcium chelator), or EGTA (5 mM) to chelate extracellular 1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin, 1 mM pervanadate, 1 mM EDTA, calcium, 20 min before infection. All the reagents were tested and found 1% Igepal, 0.25% deoxycholic acid, 1 mM NaF, and 50 mM Tris-HCl (pH not to have a significant effect on the uptake of the mycobacteria by the 7.4)). The cell lysates were removed from the plates and stored at Ϫ20°C. macrophages (data not shown). Equal amounts of protein, as defined using the Micro BCA Protein Assay 5496 MACROPHAGE SPK ACTIVATION BY MYCOBACTERIA

(Pierce), were loaded onto 10% SDS-PAGE gels, electrophoresed and pathogenic mycobacteria. To answer these questions, we looked at transferred onto polyvinylidene difluoride membrane (Millipore). The the role of SPK in macrophages infected with nonpathogenic M. membranes were blocked in TBST (Tris-buffered saline with 0.05% Tween smegmatis and pathogenic M. avium 724. Consistent with the pre- 20) supplemented with 5% powdered milk and then incubated with primary ␾ Abs against phospho-p38, phospho-ERK1/2, total ERK1/2, or phospho- vious reports, BMM infected with M. smegmatis show similar or Akt from Cell Signaling Technology. The blots were washed with TBST slightly higher levels of MAPK activation compared with macro- and incubated with a secondary Ab, either HRP-conjugated anti-rabbit or phages infected with M. avium at 30 min and 1 h postinfection anti-mouse Ig (Pierce) in TBST plus 5% powdered milk. The bound Abs (Fig. 1A). However, p38 and ERK1/2 activation is lost in macro- were detected using SuperSignal West Femto ECL reagents (Pierce). phages infected with M. avium 724 between 1 and 2 h, whereas in ELISA M. smegmatis-infected macrophages there is a sustained activation of ERK1/2 and p38 (15). Next we treated BMM␾ with SPK in- The levels of cytokines secreted by infected macrophages were measured using the commercially available ELISA reagent kits for TNF-␣ (BD hibitors DHS (24) and SKI (25) and looked at the activation of the Pharmingen), IL-6, RANTES (eBioscience), and G-CSF (R&D Systems). MAPKs in mycobacteria-infected macrophages. DHS (25 ␮M) Culture medium collected from the macrophages was analyzed for cyto- and SKI (10 ␮M) were added to the macrophages 20 min before kines according to manufacturer’s instructions and the cytokine concen- infection and did not have any effect on the uptake of mycobacteria trations were determined against the standard curves. A cytokine profile analysis was performed by using the RayBio Mouse Cytokine Ab Array by the macrophages (data not shown). Treatment with DHS and (RayBiotech) with culture supernatants from noninfected or infected mac- SKI resulted in inhibition of ERK1/2 activation in macrophages rophages (data not shown). infected with M. smegmatis or M. avium (Fig. 1, B and C). p38 Activation remained unaffected upon treatment with SPK inhibi- Statistical analysis tors. Our published studies have shown that macrophages infected Statistical significance was determined with the paired two-tailed Student’s with fast-growing, nonpathogenic mycobacteria induce signifi- t test. Values at p Ͻ 0.05 level were considered significant and determined cantly higher TNF-␣ compared with macrophages infected with using InStat/Prism software. pathogenic M. avium, and this response was dependent on MAPK Results activation (14). To investigate whether SPK is important for TNF-␣ production, we pretreated macrophages with SKIs and SPK is important for ERK1/2 activation and TNF-␣ production measured TNF-␣ secretion upon mycobacterial infection. As ob- in macrophages infected with mycobacteria served previously, M. smegmatis infection of BMM␾ resulted 2ϩ We showed previously that Ca is important for the activation of in higher levels of TNF-␣ production compared with M. avium- macrophage signaling pathways upon mycobacterial infection infected cells (Fig. 1D). This TNF-␣ secretion was blocked in (15). A study by Malik et al. (4) showed that infection with dead the presence of DHS and SKI indicating that SPK is required M. tuberculosis, but not live, leads to the activation of SPK in for the ERK1/2 activation and for TNF-␣ production in M. human macrophages. This activation of SPK was responsible for smegmatis-infected and, to a lesser extent, M. avium-infected 2ϩ the induction of Ca flux and phagosome maturation in macro- macrophages. phages 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 Ca2ϩ release (23). However, it is not PI-PLC and PKC are important in ERK1/2 activation in known whether SPK is involved in other signaling pathways upon macrophages infected with mycobacteria mycobacterial infection and whether there is a differential role for Although SPK and S1P have been linked to ERK activation in a SPK in macrophages infected with nonpathogenic compared with number of cell types, it has not been demonstrated in macrophages.

FIGURE 1. SPK is required for ERK1/2 activation and TNF-␣ production in macrophages infected with mycobacteria. A, ERK1/2 and p38 phosphor- ylation 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 (Ϫ) or DMSO (Ϫ) 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 ,Significant (p Ͻ 0.01) to control. The results are representative of three separate experiments. RC, Resting ,ء .ELISA. Values are expressed as mean ϩ SD noninfected BMM␾; Smeg, M. smegmatis; Avium, M. avium 724. The Journal of Immunology 5497

In many systems, the ERK activation is through S1P-mediated infection. To test this possibility, we measured the kinase activity activation of G protein-coupled receptors and subsequent activa- of cPKC in BMM␾ infected with M. smegmatis and M. avium 724 tion of the Ras-ERK pathway (26, 27). However, a recent study by and examined the effect of SPK and PI-PLC inhibitors on the ki- Blom et al. (28) showed that PI-PLC inhibitor U73122 blocked nase activity. We found that PKC activity is significantly higher in S1P-mediated Ca2ϩ release in HEK293 cells. Therefore, we were macrophages infected with M. smegmatis compared with M. interested in investigating the potential involvement of PLC-PKC avium-infected or noninfected macrophages at 1 h postinfection pathway in the SPK-mediated ERK1/2 activation in BMM␾ in- (Fig. 3A). BMM␾ infected with M. avium showed a slight increase fected with mycobacteria. We first tested whether PLC was in- in PKC activity above resting cells (RC, 111 Ϯ 3%), but it was not volved in ERK activation by treating infected BMM␾ with statistically significant ( p Ͼ 0.05). PKC activation induced upon U73122 (PI-PLC inhibitor) or D609 (phosphatidylcholine-specific mycobacterial infection was blocked when macrophages were pre- (PC)-PLC inhibitor). Inhibitors were added to macrophages 30 min treated with Go¨6976 (data not shown) or Ro31-8425 (Fig. 3A), postinfection and had no significant effect on phagocytosis (data demonstrating the specificity of the kinase assay for the cPKC. We not shown). As revealed in Fig. 2A, treatment with U73122 (2 saw a similar trend in PKC activation after 30 min and2hof ␮M), but not D609 (10 ␮M), results in inhibition of ERK1/2 ac- infection (data not shown). As expected, because PKC is activated tivation in macrophages infected with mycobacteria. No inhibition 2ϩ by the products of PI-PLC (i.e., DAG- and IP3-mediated Ca of p38 activation was observed. To ensure the specificity of the release), we found the PKC activation induced upon M. smegmatis inhibitor, we also treated macrophages with U73343 (2 ␮M), a infection to be inhibited by treating the BMM␾ with the PI-PLC structural analog of U73122, and found it to have no effect on inhibitor U73122 (Fig. 3B). The minimal PKC activation above ERK1/2 phosphorylation (data not shown). This result indicated RC levels observed in M. avium-infected macrophages was also that PI-PLC, but not PC-PLC, is important for ERK1/2 activation. inhibited in the presence of U73122 (from 110 Ϯ 3% to 100 Ϯ Because the products of PI-PLC activation (i.e., IP3 and DAG) 8%); however, again the levels were not statistically different. To function to stimulate cPKC, we examined the significance of these test whether SPK is present upstream of the PKC pathway, we cPKC isoforms in MAPK activation. As shown in Fig. 2, B and C, tested the effect of DHS on PKC activation. Similar to what was ERK1/2 activation in macrophages following infection with my- seen with PI-PLC inhibitors, PKC activation induced upon M. ␮ cobacteria was inhibited by Go¨6976 (1 M), a selective inhibitor smegmatis infection was inhibited in the presence of DHS (Fig. ␣ ␤ ␮ of PKC- isozyme and PKC- 1, and also by Ro31-8425 (10 M), 3C). Following an M. avium infection, PKC activation was dimin- ␣ ␤ ␤ ␥ a selective inhibitor of PKC- , I, II, and isoforms. There was ished to RC levels upon treatment with DHS (from 110 ϩ 5% to a dose-dependent inhibition of ERK1/2 activation upon treatment 98 ϩ 0.3%) (Fig. 3C). These experiments, however, did not de- with inhibitors (Fig. 2C and data not shown). p38 Activation was termine whether PI-PLC lies upstream or downstream of SPK ac- not inhibited by Go¨6976 or Ro31-8425 treatment in either M. tivation. To define where PI-PLC resides in the ERK1/2 activation smegmatis-orM. avium-infected macrophages. pathway, we infected BMM␾ in the presence of the SKI and mea-

sured IP3 levels. As shown in Fig. 3D, the addition of the SKI to PI-PLC and SPK mediate PKC activation leads to ERK1/2 M. smegmatis-infected BMM␾ lead to a significant decrease in IP ␾ 3 phosphorylation in mycobacteria-infected BMM formation, suggesting that PI-PLC activation is dependent on SPK.

We hypothesized that SPK functions through PI-PLC and PKC to M. avium infection did not lead to a significant increase in IP3 mediate ERK1/2 activation in BMM␾ following a mycobacterial levels relative to noninfected macrophages (Fig. 3D).

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 (Ϫ) 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 Go¨6976 (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␾. 5498 MACROPHAGE SPK ACTIVATION BY MYCOBACTERIA

FIGURE 3. cPKC activity is ele- vated in M. smegmatis-infected BMM␾ and is dependent on SPK and PI-PLC. A, BMM␾ infected for1horleft nonin- fected were lysed, and the cell lysates were assayed for PKC activity as de- scribed in Materials and Methods. Be- fore the PKC activity assay, one well of M. smegmatis-infected macrophages was treated with Ro31-8425 (A)asde- scribed in Fig. 2. BMM␾ were infected with M. smegmatis or M. avium with (ϩ) or without (Ϫ) U73122 (B), DHS (C), or SKI (D) as described in Figs. 1 and 2 and the cell lysates were assayed for PKC ac-

tivity (C)orIP3 levels (D). IP3 concen- trations were measured as described in Materials and Methods. Values are ex- Significant ,ء .pressed as mean ϩ SD Significant (p Ͻ ,ءء ,p Ͻ 0.05) to RC) 0.05) to Smeg (Ϫ). Data are representa- tive 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 phorylation at serine 473. This residue is a well-known substrate calcium for PI3K phosphorylation. We observed a more pronounced Akt S1P generated through SPK activity might be functioning extra- phosphorylation in macrophages infected with M. smegmatis com- pared with M. avium-infected or noninfected cells (Fig. 5A). This cellularly to stimulate IP3 formation by activating G protein-cou- pled receptors, as previously observed (7), or by working directly activation was inhibited when macrophages were treated with ␮ to stimulate PI-PLC activity. To address these possibilities, we PI3K inhibitor LY294002 (50 M). However, there was no effect performed the infection experiments in the presence of SKI or on ERK1/2 or p38 activation upon treatment with LY294002, in- DHS with or without extracellular S1P. We observed an inhibition dicating that PI3K is not present upstream of MAPK pathways. of ERK1/2 activation and TNF-␣ production in the presence of The LY294002 was added to the macrophages 30 min after infec- SPK inhibitors, and this effect was not reversed by incubation with tion due to its known effect on phagocytosis. Under these condi- S1P, suggesting that the effect of SPK on BMM␾ activation is not tions, the LY294002 did not significantly affect phagocytosis. We dependent on extracellular S1P (Fig. 4A and data not shown). The also measured Akt phosphorylation in the presence of MAPK in- hibitors. As expected, PI3K activation (measured through Akt lack of an effect by extracellular S1P and a known role for IP3 in the release of Ca2ϩ from the endoplasmic reticulum suggest that it phosphorylation) was not affected in macrophages treated with is the intracellular pool of Ca2ϩ that is released initially upon MEK1/2 inhibitor PD98059 (Fig. 5B) or p38 MAPK inhibitor III mycobacterial infection and is required for MAPK activation. (data not shown), suggesting that the PI3K is parallel to the MAPK However, to test this suggestion more directly, infection experi- pathway. We also tested whether the SPK, PI-PLC, and PKC path- ments were performed in Ca2ϩ-free medium or in the presence of ways were upstream of PI3K activation. For these experiments, we EGTA to eliminate the source of extracellular Ca2ϩ or incubated treated mycobacteria-infected BMM␾ with the various inhibitors: with BAPTA-AM to chelate intracellular Ca2ϩ. As expected, re- DHS for SPK, U73122 for PI-PLC, and Go¨6976 for PKC, and moval of Ca2ϩ from the extracellular medium did not effect looked at phosphorylation of Akt. As shown in Fig. 5, C and D, TNF-␣ secretion (Fig. 4B), MAPK activation (Fig. 4C), or IL-6 PI3K activation was inhibited in the presence of all inhibitors release (data not shown) by BMM␾ following infection. In con- placing SPK/PI-PLC/PKC upstream of PI3K. trast, chelation of intracellular Ca2ϩ 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 in- PI-PLC, PKC, and PI3K are required for TNF- production in 2ϩ macrophages infected with M. smegmatis and M. avium dicating that IP3-mediated release of intracellular Ca is required for BMM␾ activation following a mycobacterial infection stems As shown in Fig. 1C, SPK is required for TNF-␣ production in 2ϩ from studies using 2-APB, an inhibitor of IP3-induced Ca re- macrophages infected with mycobacteria. Because we found a role lease. The 2-APB inhibitor significantly blocked, in a dose-depen- for PI-PLC and PKC in ERK1/2 activation and a link between SPK dent manner, TNF-␣ production in both M. smegmatis- and M. and the PI-PLC/PKC pathway, we examined the significance of avium-infected BMM␾ (Fig. 4D). these signaling molecules in TNF-␣ production following infec- tion with M. smegmatis and M. avium. We also tested whether PI3K activity induced upon M. smegmatis and M. avium PI3K was required for TNF-␣ production because we observed its infection is dependent on SPK and PKC but not MAPK differential activation in macrophages infected with M. smegmatis PI3K has been shown to stimulate ERK1/2 activation in macro- compared with M. avium. As shown in Fig. 6A, the high TNF-␣ phages (29, 30). To determine whether PI3K is activated upon a production induced in M. smegmatis-infected macrophages was mycobacterial infection, we measured Akt/protein kinase B phos- inhibited in the presence of U73122, Go¨6976, Ro31-8425, and The Journal of Immunology 5499

␾ 2ϩ FIGURE 4. SPK-mediated activation of BMM is not dependent on extracellular S1P but is dependent on IP3-mediated release of intracellular Ca . 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 (ϩ) or without (Ϫ) 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, Ca2ϩ-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 ␮M), an inhibitor of 2ϩ ␣ ϩ IP3-induced Ca release. After infection, culture supernatants were removed and analyzed for TNF- production. Values are expressed as mean SD. Significant (p Ͻ 0.05) to control; ns, not significant (p Ͼ 0.05) to control. Data are representative of three separate experiments. RC, Resting, noninfected ,ء BMM␾; Smeg, M. smegmatis; Avium, M. avium 724.

LY294002. The low TNF-␣ production in M. avium-infected mac- 0.05). The results underscore the importance of these signaling rophages was also inhibited after treatment with the various inhib- molecules in promoting TNF-␣ production by BMM␾ upon infec- itors; however, there was only a 20–30% drop in TNF-␣ produc- tion with mycobacteria and how the differential activation of these tion with LY294002, which was not statistically significant ( p Ͼ signaling molecules could be responsible for the differences in

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 Go¨6976 (C), SPK inhibitor DHS (C), or PI-PLC inhibitor U73122 (D), or the vehicle control (Ϫ) 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␾. 5500 MACROPHAGE SPK ACTIVATION BY MYCOBACTERIA

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 differ- ent 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 ϩ SD. ns, Not significant (p Ͼ 0.05) to control M. avium. Data are representative of three separate experiments. RC, Rest- ing, noninfected BMM␾.

TNF-␣ production observed between BMM␾ infected with M. function by blocking Ca2ϩ-dependent signaling (3, 4, 32). In pri- smegmatis and with M. avium. mary human macrophages, live M. tuberculosis fails to promote SPK activity and inhibits SPK activity induced upon infection with IL-6, G-CSF, and RANTES production by Mycobacterium- heat-killed M. tuberculosis. This lack of SPK stimulation in mac- infected macrophages is dependent on the SPK/PI-PLC/PKC rophages infected with live M. tuberculosis results in a reduced pathway cytosolic Ca2ϩ concentration. This Ca2ϩ induction is necessary for To test whether activation of these signaling molecules is required phagosome maturation (4). A recent study has shown that engage- for other macrophage responses to mycobacterial infection, we ment of the mannose receptor by mannose-lipoarabinomannan, looked at the production of additional inflammatory mediators fol- which is normally expressed by M. tuberculosis, is important in lowing treatment with the different inhibitors. However, we first restricting phagosome-lysosome fusion (33). Whether there is a investigated, using a RayBio Mouse Cytokine Ab Array, whether link between engagement of the mannose receptor by M. tubercu- production of other cytokines and chemokines differed between losis and the bacilli’s ability to inhibit SPK activity warrants fur- macrophages infected with M. smegmatis or with M. avium.We ther study. found that, in addition to TNF-␣, M. smegmatis-infected macro- SPK phosphorylates a host lipid, sphingosine, to form S1P, phages showed increased levels of IL-6, G-CSF, and RANTES which is a ligand for specific G protein-coupled receptors and also compared with M. avium 724-infected cells (data not shown). A regulates intracellular Ca2ϩ homeostasis by releasing Ca2ϩ from difference in macrophage production was also observed by ELISA the cytoplasmic organelles (23, 34). SPK is activated by various (Fig. 6, B–D). Furthermore, as shown in Fig. 6, B–D, levels of all growth factors and cytokines, including FCS, platelet-derived three of these inflammatory mediators were significantly decreased growth factor (24), basic fibroblast growth factor (35), TNF-␣ in M. smegmatis-infected BMM␾ treated with the SPK, PI-PLC, (36), IL-1␤ (37), and vascular endothelial growth factor (26). and PKC inhibitors, although the PKC inhibitors Go¨6976 and Our previous studies indicated that Ca2ϩ induction was also Ro31-8425 blocked production of G-CSF and RANTES only 60– important for MAPK activation mediated through activation of 70%. However, both SPK inhibitors DHS and SKI completely or calmodulin and calmodulin kinase and that this activation was el- almost completely blocked production of all three mediators. The evated in macrophages infected with nonpathogenic mycobacteria PI3K inhibitor LY294002 completely blocked G-CSF and IL-6 but relative to cells infected with M. avium (15). However, the role inhibited RANTES production only 60–70% (Fig. 5, B–D). In M. played by SPK in the Ca2ϩ induction and MAPK activation was avium-infected BMM␾, there was a little production of G-CSF and not addressed in this study. Moreover, how SPK induces a Ca2ϩ IL-6, which were at undetectable levels in the presence of inhib- flux also was not defined in previous work. We hypothesized that itors. However, there was a significant amount of RANTES se- SPK could be involved in MAPK activation and might be respon- creted in M. avium-infected BMM␾, and it was blocked com- sible for the sustained MAPK activation and increased TNF-␣ pro- pletely by DHS and SKI, whereas LY294002, Go¨6976, and Ro31- duction seen in macrophage infected with nonpathogenic myco- 8425 resulted in 40–60% drop (Fig. 6D). bacteria. As predicted, the SPK inhibitors DHS and SKI blocked the nonpathogenic M. smegmatis and virulent M. avium 724 in- Discussion duced activation of ERK1/2 by infected macrophages. A similar Pathogenic mycobacteria successfully reside inside host macro- role for SPK has been proposed in LPS-mediated ERK1/2 activa- phages by inhibiting several host cell processes. A number of mac- tion in RAW 264.7 cells (38). This also correlates well with our rophage cellular functions have been shown to be inhibited by previous data in which we found a role for Ca2ϩ in ERK1/2 ac- pathogenic mycobacteria, including the fusion of phagosome with tivation. SPK has previously been implicated in generation of in- lysosome, Ag presentation, apoptosis, and the stimulation of anti- flammatory mediators in macrophages (39). Blocking SPK with microbial responses due to activation of pathways involving DHS and SKI also resulted in a significant drop in TNF-␣ pro- MAPKs, IFN-␥, and Ca2ϩ signaling. This modulation of host cell duction upon infection with both M. smegmatis and M. avium 724. signaling is critical for the persistence of mycobacteria within the Chelating intracellular Ca2ϩ or blocking Ca2ϩ release from the host and for the suppression of a generalized inflammatory re- endoplasmic reticulum had a similar effect on TNF-␣ production. sponse (reviewed in Refs. 12 and 31). Recently, a number of stud- This effect was more pronounced in M. smegmatis-infected ies have shown that pathogenic mycobacteria inhibit macrophages BMM␾, suggesting that SPK is required for the enhanced TNF-␣ The Journal of Immunology 5501

production observed in macrophages infected with nonpathogenic found cPKC activity to be inhibited when BMM␾ were pretreated mycobacteria. Together, these suggest that a Ca2ϩ flux following with the PI-PLC inhibitor U73122. It was also lowered to the RC a mycobacterial infection is required for optimum macrophage ac- levels upon pretreatment of macrophages with the SPK inhibitor

tivation and that SPK mediates this calcium flux. DHS. Moreover, IP3 formation, which is induced significantly in SPK promotes ERK1/2 activation in variety of cell types M. smegmatis-infected macrophages, is inhibited in the presence through various mechanisms (36, 40). In endothelial cells and tu- of SKI. These results demonstrate that SPK mediates cPKC acti- mor cells, it was shown that SPK mediates vascular endothelial vation through activation of PI-PLC. The exact mechanism of this growth factor-induced activation of Ras, Raf, and ERK1/2 by activation remains unclear; however, it does not appear to involve down-regulating Ras-GTPase-activating protein activity (26, 41). release of S1P and activation of G protein-coupled receptors be- SPK can mediate its effect as a second messenger both dependent cause the addition of extracellular S1P did not overcome the DHS- and independent of PLC pathway (reviewed in Ref. 42). S1P, the or SKI-mediated inhibition of macrophage activation. product of SPK activation, can mobilize the Ca2ϩ channels di- PKC and MAPK activation have also been closely linked to rectly or can activate the PLC-dependent pathways by activating G PI3K stimulation. PI3K is a conserved family of signaling mole- protein-coupled receptors (7). However, the mechanism by which cules that are involved in regulating cellular proliferation and sur- SPK stimulates Ca2ϩ release in macrophages under various stimuli vival. Recent data suggest that the PI3K-Akt pathway is activated has not been defined. Therefore, we tested whether the PLC-PKC in macrophages upon mycobacterial infection or treatment with signaling pathway was involved in the MAPK activation upon my- mycobacterial glycolipids (55–57). We show that PI3K activity, as cobacterial infection and whether the SPK-mediated ERK1/2 ac- defined by Akt phosphorylation, was activated at higher levels tivation involved the PLC-PKC pathway. Both PC-PLC and PI- upon infection with M. smegmatis compared with M. avium-in- PLC can stimulate MAPK activation in macrophages depending fected macrophages. PI3K activity was dependent on the SPK, on the stimulus. In human macrophages, PC-PLC, but not PI-PLC, PI-PLC, and PKC but not on ERK1/2 or p38. This finding suggests is required for LPS-induced MAPK activation (43). Interestingly, that the SPK-PLC-PKC pathway mediates ERK1/2 and PI3K ac- we found that PI-PLC, but not PC-PLC, is required for ERK1/2 tivation following infection with mycobacteria (Fig. 7). activation in macrophages upon mycobacterial infection. The What role do these pathways play in the differential activation of

products of PI-PLC activation (i.e., DAG and IP3) act as secondary macrophages following infection with nonpathogenic M. smegma- messengers for the activation of cPKC isoforms. Therefore, we tis compared with pathogenic M. avium? To address this question, investigated activation of cPKC upon infection with mycobacteria. we assayed for various inflammatory mediators upon infection PKC was originally described as a Ca2ϩ- and phospholipid- with the mycobacteria in the presence or absence of the different dependent protein kinase activated by DAG and other lipids. The inhibitors. Previous studies have implicated PLC-PKC in the pro- ␣ ␤ ␣ PKC family is divided into three groups: 1) cPKCs comprise the , duction of IL-1 , TNF- , NO, and PGE2 by macrophages upon ␤ ␤ ␥ 2ϩ ␥ I, II, and isoforms and are dependent on Ca and DAG; 2) LPS treatment and Fc R stimulation (20, 51, 58–60). In J774.1 novel PKCs, including the PKC-␦, ⑀, ␩, and ␪ isoforms, are Ca2ϩ macrophages, PC-PLC, but not PI-PLC, is required for LPS-in- independent and regulated by DAG and phosphatidylserine; and 3) duced inducible NO synthase expression (61). In addition, prior atypical PKCs, including PKC-␨ and PKC-␭ isoforms, are Ca2ϩ studies have shown that inhibition of PLC-PKC and PI3K block and DAG independent (44, 45). PKCs have been shown to be macrophage production of IL-8, MIP-2, NO, and MCP-1 following activated during phagocytosis and play an important role in phago- a mycobacteria infection (55, 62). However, no study has ad- some maturation and immune signaling (46–49). Some PKC iso- dressed the role of SPK in macrophage activation. As shown ear- forms are implicated in Fc␥R-mediated phagocytosis but cPKCs lier, BMM␾ infected with M. smegmatis induced significantly have been shown not to be required for phagocytosis (50). As higher production of TNF-␣, G-CSF, IL-6, and RANTES com- expected, we found a similar effect of cPKC inhibitors on ERK1/2 pared with M. avium-infected cells. Moreover, production of these activation as seen with the PI-PLC and SPK inhibitors. Our data mediators was blocked significantly upon treatment of macro- showing a lack of ERK1/2 activation in BMM␾ treated with cPKC phages with the PI-PLC and PKC inhibitors and to a lesser extent inhibitors Go¨6976 and Ro31-8425 correlated well with other stud- with the PI3K inhibitor. As expected, based on our signaling ex- ies in which cPKC was required for LPS induced ERK1/2 activa- periments, inhibiting SPK activity blocked production of these in- tion in macrophages (19, 51). However, p38 activation was not flammatory mediators with the greatest decrease associated with affected by cPKC inhibition, perhaps due to the regulation of p38 M. smegmatis-infected macrophages. The inhibitors also blocked by other isoforms of PKC, as shown previously with LPS-induced the limited production of these inflammatory mediators following JNK but not p38 activation, being dependent on PKC-⑀ (52). Also a M. avium infection although to different extents. As mentioned as predicated, we found PKC activity to be elevated in macro- previously, the inhibitors also blocked the M. avium-induced phages infected with M. smegmatis compared with uninfected or ERK1/2 activation. In contrast, we did not observe significantly ␾ ␾ M. avium-infected BMM . This cPKC activity was blocked upon higher levels of IP3 formation or PKC activity in BMM infected pretreating macrophages with Go¨6976 or Ro31-8425 confirming with M. avium compared with uninfected cells. This apparent con- the specificity of the assay for cPKCs. The high background of tradiction might be explained by a limitation in PI-PLC and PKC cPKC activity in noninfected BMM␾ likely results from the ki- detection assays. We hypothesize that M. avium induces only a nases’ role in maintaining cellular adhesion. It also suggests that limited activation of the SPK/PI-PLC/PKC pathway, which is not the increase in cPKC activity is required for the prolonged ERK1/2 detectable above the background measured in noninfected cells. activation seen in M. smegmatis-infected macrophages (i.e., 2 and Nevertheless, this limited activation is required for ERK1/2 phos- 4 h postinfection). To our knowledge this is the first report show- phorylation and for the induction of a macrophage proinflamma- ing differential activation of cPKC upon infection with pathogenic tory response. and nonpathogenic mycobacteria. A recent study indicated a sim- Previously, we showed that M. smegmatis infection leads to in- ilar activation of PKC-␣ by noncapsulated mutant Streptococcus creased activation of the calmodulin/calmodulin kinase and suis but not by pathogenic encapsulated bacteria (53). cPKC have cAMP-protein kinase A pathways, which are also required for also been shown to be activated in macrophages upon stimulation ERK1/2 activation and up-regulation of TNF-␣ production in with cord factor from M. tuberculosis (54). In our studies, we BMM␾ (15). We extend these findings in the present study by 5502 MACROPHAGE SPK ACTIVATION BY MYCOBACTERIA

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