p38 MAPK Autophosphorylation Drives Macrophage IL-12 Production during Intracellular Infection

This information is current as Leesun Kim, Laura Del Rio, Barbara A. Butcher, Trine H. of September 28, 2021. Mogensen, Søren R. Paludan, Richard A. Flavell and Eric Y. Denkers J Immunol 2005; 174:4178-4184; ; doi: 10.4049/jimmunol.174.7.4178

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

p38 MAPK Autophosphorylation Drives Macrophage IL-12 Production during Intracellular Infection1

Leesun Kim,* Laura Del Rio,* Barbara A. Butcher,* Trine H. Mogensen,† Søren R. Paludan,† Richard A. Flavell,‡ and Eric Y. Denkers2*

The intracellular protozoan Toxoplasma gondii triggers rapid MAPK activation in mouse macrophages (M␾). We used synthetic inhibitors and dominant-negative M␾ mutants to demonstrate that T. gondii triggers IL-12 production in dependence upon p38 MAPK. Chemical inhibition of stress-activated protein kinase/JNK showed that this MAPK was also required for parasite- triggered IL-12 production. Examination of upstream MAPK kinases (MKK) 3, 4, and 6 that function as p38 MAPK activating kinases revealed that parasite infection activates only MKK3. Nevertheless, in MKK3؊/؊ M␾, p38 MAPK activation was near normal and IL-12 production was unaffected. Recently, MKK-independent p38␣ MAPK activation via autophosphorylation was

described. Autophosphorylation depends upon p38␣ MAPK association with adaptor protein, TGF-␤-activated protein kinase Downloaded from 1-binding protein-1. We observed TGF-␤-activated protein kinase 1-binding protein-1-p38␣ MAPK association that closely par- alleled p38 MAPK phosphorylation during Toxoplasma infection of M␾. Furthermore, a synthetic p38 catalytic-site inhibitor blocked tachyzoite-induced p38␣ MAPK phosphorylation. These data are the first to demonstrate p38 MAPK autophosphory- lation triggered by intracellular infection. The Journal of Immunology, 2005, 174: 4178–4184.

roduction of IL-12 is pivotal in generation of Th1 T lym- be the critical molecules in driving NF-␬B-dependent IL-12(p40) http://www.jimmunol.org/ phocyte effectors that are required for resistance to many transcription, yet Toxoplasma drives normal production of this cy- P microbial pathogens. This principle is well illustrated in tokine in c-RelϪ/Ϫ cells (10, 11). Furthermore, recent evidence the case of the intracellular protozoan Toxoplasma gondii, a com- suggests T. gondii actively subverts NF-␬B activation during in- mon opportunistic parasite causing asymptomatic infection in nor- fection (12). mal hosts, but a pathogen that is uniformly lethal in the absence of The C/EBP proteins are under the control of CREB, a down- host IL-12 (1). Cells such as macrophages (M␾),3 dendritic cells, stream target of MAPK (13, 14). The MAPK signal transduction and neutrophils produce IL-12 in response to T. gondii (2–5). pathways are highly conserved cascades important in diverse as- TLRs are implicated in recognition of the parasite, because IL-12 pects of the immune response (15). They center on a family of by guest on September 28, 2021 production is defective in MyD88-deficient mice, and this is as- protein kinases that include stress-activated protein kinase/JNK ␥ sociated with acute susceptibility to infection (6). The IFN- con- (SAPK/JNK), ERK, and p38. The MAPK may be present in both sensus sequence binding protein is one transcription factor shown cytosolic and nuclear compartments, where they phosphorylate ␾ to be involved in M IL-12 production in response to T. gondii (7). transcription factors such as Elk-1, C/EBP␤, activating transcrip- Beyond this, little is known regarding IL-12 induction pathways tion factor (ATF)-2, and c-Jun upon activation. MAPK may also during Toxoplasma infection. activate other protein kinases such as MAPK-activated protein ki- The IL-12(p40) promoter contains control elements that bind nase (MAPKAPK) 2, which is a substrate for p38 (16). ␬ NF- B/Rel and C/EBP transcription factors (8, 9). The role of MAPK are activated by dual phosphorylation mediated by up- NF-␬B signaling in IL-12 induction during T. gondii infection is stream MAPK kinases (MKK). Studies with small molecule in- uncertain. This is because c-Rel/p50 heterodimers are believed to Ϫ Ϫ hibitors of p38, as well as MKK3 / mice that lack a major up- stream kinase of p38, have implicated the p38 signaling cascade in M␾ TNF-␣ and IL-12 production during LPS stimulation (17–20). *Department of Microbiology and Immunology, College of Veterinary Medicine, The MKK in turn are activated by a large family of MKK kinases Cornell University, Ithaca, NY 14853; †Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark; and ‡Department of Immu- (M3K), a prominent one of which is TGF-␤-activated protein ki- nology and Howard Hughes Medical Institute, Yale University School of Medicine, nase 1 (TAK1), which has been shown to lead to activation of p38 New Haven, CT 06520 and SAPK/JNK, as well as the I␬B kinase complex leading to Received for publication August 23, 2004. Accepted for publication December ␬ 30, 2004. NF- B activation (21, 22). TAK1 associates with TAK1-binding protein (TAB)1 and TAB2. Activation of the TAK1 complex relies 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 upon ubiquitination of TNFR-associated factor 6 (23). The TNFR- with 18 U.S.C. Section 1734 solely to indicate this fact. associated factor 6 molecule itself is linked to the TLR system, and 1 This work was supported by National Institute of Allergies and Infectious Diseases this occurs through interactions with IL-1R-associated kinase 1 Grant AI 50617. and 4, and adaptor protein MyD88 (24). 2 Address correspondence and reprint requests to Dr. Eric Y. Denkers, Department of We and others recently showed that tachyzoite infection of mu- Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. E-mail address: [email protected] rine M␾ triggers activation followed by deactivation of the three 3 Abbreviations used in this paper: M␾, macrophage; MKK, MAPK kinase; KO, major MAPK (25, 26). Here, we use pharmacological inhibitors knockout; SAPK, stress-activated protein kinase; WT, wild type; ATF, activating and dominant-negative mutants to demonstrate a requirement for transcription factor; MAPKAPK, MAPK-activated protein kinase; TAK1, TGF-␤- ␾ activated protein kinase 1; TAB, TAK1-binding protein; STAg, soluble tachyzoite p38 in IL-12 production by Toxoplasma-infected M . Phosphor- Ag. ylation of this MAPK could not be attributed to the activity of

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 4179

MKK3, 4, or 6 that are known to be the upstream p38-activating harvested, 1.6 ml of ice-cold cell lysis buffer (20 mM Tris-HCl (pH 7.5), MKK. Instead, we show that T. gondii, in contrast to LPS, triggers 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM ␤ sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM Na3VO4,1 a recently described pathway of activation involving TAB1-depen- ␮ ␣ g/ml leupeptin, and 1 mM PMSF) was added and samples were boiled for dent autophosphorylation of p38 (27–29). Our results are the first 10 min. The supernatants were incubated with rabbit anti-p38␣ Ab (Cell to show the importance of p38 autoactivation during microbial Signaling Technology) with gentle rocking for 18 h at 4°C. Lysates were infection. subsequently incubated with protein A-Sepharose for an additional3hat 4°C. Then p38␣ immunoprecipitates were washed three times with lysis buffer and once with ice-cold PBS. Samples were resuspended in reducing Materials and Methods SDS sample buffer, boiled (3 min), and then subjected to immunoblot Mice and parasites analysis for phospho- and total p38. To detect p38 association with TAB1, M␾ were infected with T. gondii Female C57BL/6 strain mice (6–8 wk of age) were obtained from Taconic Ϫ Ϫ Ϫ Ϫ or stimulated with LPS, then cells were washed with ice-cold PBS and Farms and JNK2 / and TLR2 / mice were purchased from The Jackson Ϫ Ϫ incubated 10 min in ice-cold lysis buffer consisting of 20 mM Tris-HCl (pH Laboratory. The targeting strategies for MKK3 / mice have been de- Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 scribed previously (18). Colonies of JNK2 / , TLR2 / , and MKK3 / mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM Na VO 1 animals were established in the Cornell University College of Veterinary 3 4, ␮g/ml leupeptin, and 1 mM PMSF. Detergent extracts were clarified by Medicine animal facility. RH strain tachyzoites of T. gondii were main- centrifugation at 18,000 ϫ g for 10 min at 4°C. The resulting supernatants tained in human foreskin fibroblast monolayers by biweekly passage in were incubated with immobilized mouse anti-phospho-p38 Ab (Cell Sig- DMEM (Invitrogen Life Technologies) supplemented with 1% heat-inac- naling Technology) with gentle rocking for 18 h at 4°C. Then phospho-p38 tivated FCS (HyClone), 100 U/ml penicillin (Invitrogen Life Technolo- immunoprecipitates were washed three times with lysis buffer and once gies), and 0.1 mg/ml streptomycin (Invitrogen Life Technologies). Parasite with ice-cold PBS. Samples were resuspended in reducing SDS sample cultures were tested every 4–6 wk for Mycoplasma contamination using a buffer, boiled (3 min), then subjected to immunoblot analysis. Downloaded from commercial PCR-ELISA-based kit (Roche Applied Science). Soluble tachyzoite Ag (STAg) was prepared from tachyzoites of T. gondii strain Western blotting RH as previously described (30). Immunoblot analysis was performed as previously described (25). Reagents Cytokine ELISA The rabbit anti-phospho-specific p38, SAPK/JNK, ERK1/2, MAPKAPK2, MKK3/6, MKK4, and c-Jun Ab, and rabbit anti-p38␣ Ab were purchased Production of IL-12(p40) was measured by cytokine ELISA as described http://www.jimmunol.org/ from Cell Signaling Technology, mouse anti-p30 (SAG-1) Ab was ob- previously (32). tained from Argene, and rabbit anti-p38 Ab as well as goat anti-TAB1 Ab were obtained from Santa Cruz Biotechnology. The inhibitors SB202190 Statistics and SB203580 were from Calbiochem, U0126 was from Cell Signaling The statistical significance of the data was determined by unpaired Stu- Technology, and SP600125 was purchased from Biomol Research Labo- dent’s t test. Values of p Ͻ 0.05 were considered significant. All experi- ratories. Ultra-pure LPS from Salmonella minnesota R595 was purchased ments were performed three or more times. from List Biological Laboratories. Anisomycin was obtained from Sigma- Aldrich and protein A-Sepharose was purchased from Amersham Results Biosciences. Toxoplasma-induced IL-12 requires activation of MAPK

Cell culture signaling components by guest on September 28, 2021 Bone marrow-derived M␾, prepared as described previously (25), and mu- We examined activation of MAPK signaling components during rine M␾ cell lines were cultured in DMEM supplemented with 10% FCS, infection of bone marrow-derived M␾ by immunofluorescence mi- ␮ penicillin (100 U/ml), streptomycin (100 g/ml), and 3% HEPES (1 M). croscopy (Fig. 1). Infected cells displayed phosphorylated p38 For in vitro infection, RH strain tachyzoites or LPS were added to M␾ in plates (Costar, Corning, NY) and plates were briefly centrifuged (3 min, (Fig. 1, A and G), SAPK/JNK (B and H) and ERK1/2 (C and I) that 200 ϫ g) to synchronize contact between M␾ and parasites. Samples were were distributed throughout the cytoplasm and nuclei. In contrast, subsequently collected for biochemical analyses or cytokine ELISA. minimal levels of these MAPK were evident in noninfected cells Immunofluorescence microscopy (Fig. 1, M–O). We also found translocation of the MAPK sub- strates c-Jun (D and J), ATF-2 (E and K), and MAPKAPK2 (F and Bone marrow-derived M␾ were allowed to adhere on sterile 12-mm cov- L) in infected M␾, unlike in noninfected cells (P–R). erslips in 24-well plates for 3 h. After 30 min of T. gondii infection (1:3 ratio of cells to parasites), M␾ were fixed with 3% paraformaldehyde for We used pharmacological inhibitors to examine MAPK activa- 10 min and permeabilized and blocked simultaneously with 5% normal tion requirements in parasite-induced IL-12 production. As shown goat serum (Jackson ImmunoResearch Laboratories) in PBS with 0.075% in Fig. 2A, SB202190 and SP600125, inhibitors respectively of saponin (Sigma-Aldrich). After overnight incubation at 4°C with rabbit p38 and SAPK/JNK kinase activities, prevented T. gondii-trig- primary Ab, mouse anti-p30 Ab was added for an additional 40 min at gered production of IL-12(p40). In contrast, blocking ERK1/2 room temperature. After washing, cells were incubated for 45 min with Alexa Fluor 594, labeled goat anti-rabbit, or Alexa Fluor 488-conjugated phosphorylation with U0126 resulted in a slight increase in IL-12. goat anti-mouse Ab (Molecular Probes). Nuclei were stained with 4Ј,6Ј- The latter result is consistent with reports suggesting a role for diamidino-2-phenylindole (Kirkegaard and Perry Laboratories) for the last ERK1/2 in negatively regulating signaling pathways leading to 10 min of incubation in secondary Ab. After washing, coverslips were IL-12 synthesis (33, 34). Phospho-specific Western blots (Fig. 2B) mounted with ProLong Antifade reagent (Molecular Probes). Images were collected with a BX51 fluorescence microscope (Olympus) equipped with demonstrated the effectiveness of each inhibitor in blocking the a DP 70 camera using DP controller software (version 1.1.1.65; Olympus) activities of p38, SAPK/JNK, and ERK1/2. We also confirmed the and DP manager software (version 1.1.1.71; Olympus). specificity of each inhibitor using phospho-specific immunoblot- ting (data not shown). Stable transfectants Recent evidence suggests that the p38 MAPK inhibitor Dominant-negative mutants of p38 and TAK1 were generated by stable SB202190 displays inhibitory effects on tachyzoite replication ␾ transfection of J774A.1 murine M as described previously (31). (35). To avoid this potential adverse effect, we used a dominant- Immunoprecipitation negative p38 M␾ cell line (dnp38) to assess the requirement for host p38 in parasite-induced IL-12 production. The parental To detect phosphorylated p38␣, wild-type (WT) and MKK3Ϫ/Ϫ M␾ were infected with T. gondii or stimulated with LPS, cells were washed with J774A.1 line produced significantly less IL-12 than bone marrow- PBS, then 0.4 ml of denaturing cell lysis buffer (50 mM Tris-HCl (pH 7.5), derived M␾ following infection (Fig. 2, A vs C). Nevertheless, 70 mM 2-ME, and 1% SDS) was added. Immediately after, cells were parasite-induced production of the cytokine was totally obliterated 4180 IL-12 INDUCTION PATHWAY IN Toxoplasma INFECTION

FIGURE 1. Activation of MAPK signaling components during T. gondii infection. Bone marrow-derived M␾ were infected with RH strain tachyzoites (A–L) or left uninfected (M–R). For infected cells, 30 min after tachyzoite addition, cells were fixed and stained with Ab to Toxoplasma and phospho-p38 (A, G, and M), phospho-SAPK/JNK (B, H, and N), phospho-ERK1/2 (C, I, and O), phospho-c-Jun (D, J, and P), phospho-ATF-2 (E, K, and Q), and phospho-MAPKAPK2 (F, L, and R). Parasites were visualized with mAb to SAG-1 (tachyzoite surface Ag-1) and Alexa Fluor 488-conjugated secondary Ab (green). MAPK signaling components were stained with Alexa Fluor 594-labeled secondary Ab (red). Cell nuclei were stained with 4Ј,6Ј-diamidino- Downloaded from 2-phenylindole (blue). The fields in G–L are shown by differential interference contrast imaging of areas in A–F. After infection, phosphorylated p38, SAPK/JNK, and ERK1/2 are distributed throughout the cytoplasm and nuclei (A–C). Activated c-Jun, ATF2, and MAPKAPK2 display strong nuclear localization after infection (D–F). Tachyzoites cross-react with the anti-ERK1/2 and anti-MAPKAPK2 Ab and therefore appear yellow (C and F). The scale bar (in R) denotes 5 ␮m. http://www.jimmunol.org/ in dnp38 cells (Fig. 2C). A similar p38 requirement was also found tivation, Toxoplasma does not induce phosphorylation of during stimulation with LPS (Fig. 2D). We used M␾ from this MKK. JNK2Ϫ/Ϫ mice to evaluate the role of this SAPK/JNK molecule in T. gondii induced IL-12 levels in MKK3-deficient M␾ that were IL-12 production. Absence of functional JNK2 had no effect on not significantly different from WT cells (Fig. 4B). In line with IL-12 synthesis induced during Toxoplasma infection. In contrast, previously published results, LPS-driven IL-12 production was de- LPS-triggered IL-12 release displayed a partial dependence upon creased by ϳ50% in the absence of MKK3 (18). Taken together, JNK2. Taken together, the results show that T. gondii triggers M␾ LPS-induced p38 phosphorylation that is required for IL-12 pro- by guest on September 28, 2021 IL-12 production through p38 and SAPK/JNK signaling pathway. duction (25) is likely to involve the combined activities of MKK3 LPS- but not Toxoplasma-induced IL-12 is partially and MKK4. In contrast, while p38 activation is required for par- JNK2-dependent. asite-induced IL-12, p38 phosphorylation can proceed in the ab- Although LPS stimulation and T. gondii infection both provide sence of activated MKK3, 4, or 6. a stimulus for IL-12 synthesis, the cytokine is produced with dis- tinct kinetics in each case. As little as 6 h was required to induce maximal IL-12 levels during LPS stimulation (Fig. 3A). In con- TAK1 is not required for Toxoplasma-induced p38 activation trast, parasite-induced IL-12 required 40 h to reach maximal lev- but is necessary for parasite-triggered IL-12 production els. We recently reported that a soluble preparation of tachyzoite The TAK1 molecule is a M3K family member implicated in ac- lysate (STAg) triggered M␾ IL-12 production through TLR2 (30). tivation of the I␬B kinase complex and MKK proteins (22, 36). We Here, we show alleviation of this requirement during live infection used a dominant-negative approach to determine the role of TAK1 (Fig. 3B). Nevertheless, there was a small reduction in IL-12 levels in parasite vs endotoxin-mediated p38 activation and IL-12 pro- in Toxoplasma-infected TLR2 knockout (KO) M␾, suggesting par- duction. Fig. 5A shows that Toxoplasma activates p38 normally in tial involvement of TLR2 in this situation. dnTAK1 M␾. Nevertheless, parasite-induced activation of MKK3, detected using an anti-phospho-MKK3/6 Ab, was defective in the T. gondii induces p38 phosphorylation in the absence of dominant-negative TAK1 line. As with bone marrow-derived M␾ activated MKK3, MKK4, and MKK6 (Fig. 4), tachyzoite infection failed to elicit MKK4 activation in The p38 MAPK is phosphorylated by upstream kinases MKK3 and parent or dnTAK1 J774A.1 cells. The effects of LPS stimulation 6, as well as MKK4 (15). We used KO mice to examine role of were distinct. Here, p38 was activated in the dnTAK1 M␾ to a MKK3, a major activator of p38, in parasite-induced p38 phos- lesser extent and with delayed kinetics relative to parental J774A.1 phorylation and IL-12 production. Even in MKK3Ϫ/Ϫ M␾, both cells (Fig. 5A). This activity was paralleled by delayed MKK3 tachyzoites and LPS induced rapid p38 phosphorylation that was phosphorylation during LPS stimulation of the dominant-negative only marginally less than that obtained using WT cells (Fig. 4A). TAK1 line. In contrast, there was an almost complete failure of Using an Ab reactive with phosphorylated forms of both MKK3 MKK4 activation in dnTAK1 M␾. and MKK6, we found no evidence for MKK6 activation in re- Despite the fact that tachyzoites induced p38 activation in sponse to T. gondii or LPS, and as expected, MKK3 activation dnTAK1 M␾, IL-12 production was reduced to background levels only in WT M␾ (Fig. 4A). This contrasted with anisomycin treat- (Fig. 5B). This is presumably an indication that other TAK1-de- ment that induced strong phosphorylation of both MKK3 and 6 in pendent kinases are required for parasite-induced IL-12 produc- WT cells, and MKK6 activation only in KO M␾. Fig. 4A also tion. The production of IL-12 triggered by LPS was defective in shows that while both LPS and anisomycin stimulate MKK4 ac- the dnTAK1 cells (Fig. 5C). Together, the results shown in Figs. 4 The Journal of Immunology 4181 Downloaded from

FIGURE 3. Control of IL-12 production in T. gondii and LPS-stimu- lated M␾. A, Kinetics of IL-12 production. Bone marrow-derived M␾ were either infected with Toxoplasma (Tg; 3:1 ratio of parasites to cells) or stimulated with LPS (100 ng/ml). At the indicated time points, supernatants were collected for IL-12 ELISA. Cells cultured in medium alone released undetectable amounts of IL-12(p40) at each time point examined (data not http://www.jimmunol.org/ shown). B, Function of TLR2 in parasite-induced IL-12. M␾ from WT and KO animals were incubated with STAg (10 and 100 ␮g/ml), RH strain tachyzoites (Tg; 1:1 and 3:1 ratio of parasites to cells), LPS (5 and 50 ng/ml), or medium alone (Med). Supernatants were collected 36 h later for .p Ͻ 0.05 comparing WT and KO ,ء .cytokine ELISA

FIGURE 2. Both p38 and SAPK/JNK MAPK are required for Toxo- association with TAB1 (28). We sought to determine whether T. plasma-induced M␾ IL-12 production. A, Use of pharmacological inhibi- gondii used this p38 activation pathway, making use of the phar- by guest on September 28, 2021 tors to probe MAPK requirements for IL-12 induction during infection. macological inhibitor SB203580. This molecule blocks p38␣ ki- Bone marrow-derived M␾ (4 ϫ 105) were precultured 2 h with p38 inhib- nase activity, but does not prevent p38␣ transphosphorylation me- itor (SB202190, 10 ␮M), SAPK/JNK inhibitor (SP600125, 20 ␮M), diated by upstream MKK (37). Accordingly, we infected or LPS- ERK1/2 inhibitor (U0126, 20 ␮M), or with the equivalent concentration of stimulated WT and MKK3 KO M␾ in the presence and absence of solvent alone (DMSO). Cells were infected with RH strain T. gondii (Tg; SB203580, then immunoprecipitated p38␣ MAPK and immuno- 3:1 ratio of parasites to cells) or left in medium alone (Med), then 36 h blotted with phospho-specific p38 Ab. Fig. 6A shows that catalyt- later, supernatants were collected for cytokine ELISA. B, Phospho-specific ic-site inhibition of p38 prevents tachyzoite-induced p38 MAPK Western blot analysis confirms effectiveness of MAPK inhibitors. Cells ϩ/ϩ Ϫ/Ϫ (2 ϫ 106) were pretreated under the conditions in A, stimulated with LPS activation in both MKK3 and MKK3 cells, yet has minimal (100 ng/ml), and samples were collected for immunoblot analysis at the effect on LPS-induced p38 phosphorylation. Control blots using indicated times. SB202190 and SP600125 target catalytic rather than phos- Ab against total p38 MAPK confirmed presence of the ␣ isoform phorylation sites on each respective MAPK. MAPKAPK2 and c-Jun are in immunoprecipitates from infected M␾ (Fig. 6A). substrates of p38 and SAPK/JNK, respectively. U0126 prevents ERK1/2 We then asked whether Toxoplasma infection triggered interac- phosphorylation through inactivation of upstream MKK (MEK1/2). C, Par- tion between p38 and TAB1, an association predicted for p38 ac- asite-induced IL-12 release fails to occur in cells expressing a dominant- tivation through autophosphorylation. Accordingly, M␾ were in- ␾ ϫ 6 negative p38 form. Parental J774A.1 and dnp38 M (1 10 ) were in- fected with T. gondii or stimulated by LPS, then p38 was fected with T. gondii (Tg; 3:1 ratio of parasites to cells) then supernatants immunoprecipitated using phospho-specific Ab. Immunoprecipi- were collected for IL-12 ELISA 48 h later. D, As in C, using LPS (100 Ϫ Ϫ tates were subsequently analyzed by Western blotting with anti- ng/ml) to stimulate cells. E, Bone marrow-derived WT and JNK2 / M␾ (4 ϫ 105) were infected with the indicated amounts of tachyzoites, and TAB1 and anti-p38 Ab. Fig. 6C demonstrates that TAB1 copre- p Ͻ 0.01 cipitates with p38 in lysates from tachyzoite-infected cells. In ,ء .supernatants were collected 36 h later for cytokine ELISA comparing WT and KO at each LPS dose. F, As in E, using LPS (100 contrast, during LPS stimulation there was no evidence for TAB1 ng/ml). association with p38, despite the fact that the total amount of active p38 during LPS stimulation exceeded that occurring during Tox- oplasma infection (Fig. 6C). Together, the results support a model and 5 argue that parasite-triggered p38 activation can proceed in which T. gondii induces M␾ IL-12 production using a mecha- without activation of MKK3, 4, or 6. nism involving TAB1-dependent p38 autoactivation, bypassing conventional MKK-driven activation of this MAPK. Toxoplasma, but not LPS, induces p38 autophosphorylation Recently, an alternative p38 activation mechanism that bypasses Discussion MKK-dependent MAPK phosphorylation was described. This in- The MAPK signaling cascades are important in both innate and volves autophosphorylation of the p38␣ isomer dependent upon acquired host defense mechanisms (15). Here, we show that p38 is 4182 IL-12 INDUCTION PATHWAY IN Toxoplasma INFECTION

FIGURE 4. Role of MKK in Toxoplasma-induced p38 phosphorylation and IL-12 production. A, Phos- phorylation of p38 and upstream MKK in WT and MKK3 KO M␾. Bone marrow-derived cells (2 ϫ 106) were incubated with T. gondii (Tg; 6:1 ratio of parasites to cells), LPS (100 ng/ml), and anisomycin (25 ␮g/ml), then at the indicated time points, lysates were prepared for Western blot analysis using Ab specific for phospho- p38, phospho-MKK3/6, phospho-MKK4, and total p38 as a protein loading control. B, Parasite-triggered IL-12 in the absence of MKK3. Bone marrow-derived M␾ from WT and MKK3 KO mice were infected with Tox- oplasma (Tg; 3:1 ratio of parasites to cells), and 36 h later supernatants were harvested for IL-12 ELISA. Med, cells cultured in medium alone. C, As in B, using Downloaded from LPS (100 ng/ml). http://www.jimmunol.org/ essential for IL-12 induction by the intracellular microbial patho- ined (38). Live parasite-induced p38 activation most likely in- gen T. gondii. Recently, soluble Toxoplasma extracts were also volves a recently described mechanism involving TAB1-mediated shown to induce M␾ IL-12 in dependence upon p38 MAPK, al- autophosphorylation rather than MKK-mediated transphosphory- though in that case involvement of upstream MKK was not exam- lation (28, 29). As such, our data are the first to provide evidence that p38 autophosphorylation is an important activation pathway during microbial infection. Activation of MAPK involves dual phosphorylation of threo- nine and tyrosine residues on a conserved regulatory TXY loop. by guest on September 28, 2021 Upstream MKK3 and MKK6 are important in p38 activation (36, 39–45). In addition, MKK4, a member of the SAPK/JNK signal- ing module, activates p38 under some conditions (46, 47). Here,

FIGURE 6. Evidence for p38 autophosphorylation during Toxoplasma infection. A, Catalytic-site inhibitor SB203580 blocks parasite-induced p38␣ phosphorylation. Bone marrow-derived WT and MKK3Ϫ/Ϫ M␾ were preincubated 30 min in the presence of SB203580 (2 ␮M) or in solvent FIGURE 5. Role of TAK1 in p38 activation and IL-12 production. A, alone (DMSO), then infected (Tg; 6:1 ratio of tachyzoites to cells) or LPS Activation of p38 in the absence of functional TAK1. J774A.1 M␾ and stimulated (100 ng/ml). At 10 and 20 min postinfection and 10 min post- dominant-negative TAK1 cells derived from J774A.1 (dnTAK1) were in- stimulation, cell lysates were collected and subjected to immunoprecipita- fected with T. gondii (Tg; 6:1 ratio of parasites to cells) or stimulated with tion with anti-p38␣ Ab. The precipitates were subsequently probed by LPS (100 ng/ml), and cell lysates were prepared at the indicated times. immunoblotting with Ab to phospho-p38 and reprobed with Ab to total Activation of p38, MKK3/6, and MKK4 was determined with phospho- p38. B, Association of TAB1 with p38 in parasite infected, but not LPS- specific Ab, and equivalent sample loading was confirmed using Ab to total stimulated cells. Bone marrow-derived M␾ were T. gondii infected (Tg; p38. B, Requirement for TAK1 in Toxoplasma-driven IL-12 production. 6:1 ratio of parasites to cells) or LPS stimulated (100 ng/ml). Samples were Parent and dnTAK1 cells were infected with T. gondii (Tg; 3:1 ratio of collected at the indicated time points and subjected to p38 immunoprecipi- parasites to cells) or cultured in medium (Med), then supernatants were tation using phospho-specific Ab. Precipitates were examined by Western collected 48 h later for IL-12 ELISA. C, As in B, using LPS (100 ng/ml). blot analysis using anti-TAB1 and anti-p38 Ab. The Journal of Immunology 4183

LPS stimulation led to partially reduced p38 activation and de- Previous results have indicated Toxoplasma-induced inhibition creased IL-12 production in MKK3Ϫ/Ϫ bone marrow-derived M␾, of LPS-stimulated IL-12 and TNF-␣ that may relate to defects in results that are in line with previously published findings (18). In endotoxin-triggered NF␬B activation and MAPK activation (25, contrast to anisomycin stimulation, neither LPS triggering nor Tox- 32, 53). Nevertheless, as shown here, M␾ eventually produce oplasma infection induced significant MKK6 activation in WT or IL-12 during T. gondii infection. Therefore, it seems likely that KO cells. For LPS stimulation, p38 phosphorylation may therefore Toxoplasma prevents proinflammatory cascades triggered by en- occur through the combined activities of MKK3 and MKK4. The dotoxin, while simultaneously eliciting IL-12 through its own inability of T. gondii to activate MKK4 to detectable levels argues unique, partially overlapping transduction pathways. The finding against this particular MKK in parasite-induced p38 activation. that TNF-␣ remains potently suppressed at all time points during Recently, MKK-independent p38 phosphorylation was de- M␾ infection suggests that this cytokine, and not IL-12, may be scribed (28, 29, 48). This involves the scaffold protein TAB1 that the true target of inhibition during Toxoplasma infection (54). normally functions in TAK1 activation (22, 49). The TAB1 mol- Production of IL-12 in T. gondii infected M␾ occurred at lower ecule can associate with the p38␣ isomer, inducing autophosphor- levels and with distinct kinetics relative to LPS. Maximal amounts ylation and activation of the latter. Although the molecular mech- of IL-12 were achieved within6hofendotoxin stimulation. In anism involved is not yet established, TAK1 and p38␣ binding contrast, parasite-induced IL-12 was not detected before 18 h and involves distinct TAB1 domains, as shown by the recent identifi- did not reach peak levels until 40 h postinfection. The delay in cation of a TAB1 splice variant that binds and activates p38␣ but tachyzoite-elicited IL-12 could reflect a requirement for de novo not TAK1 (29). TAB1-mediated p38 autophosphorylation is pro- synthesis of transcription factors driving IL-12 synthesis. In this posed as a mechanism involved in maintaining basal p38 activa- regard, p38 is involved in CREB phosphorylation, the latter in turn Downloaded from ␤ tion levels, as well as contributing to activation through surface increasing C/EBP transcription that is proposed as a factor for ␤ receptors. In addition, p38 autophosphorylation and activation has IL-12(p40) gene induction (13, 14). Increased C/EBP transcrip- been implicated in a model of myocardial injury during ischemia tion only occurs after 2–4 h of LPS stimulation, and therefore and reperfusion (27). other factors are likely involved in early endotoxin-induced IL-12 Data that led us to examine Toxoplasma-induced p38 autophos- production (55). For Toxoplasma, a possible scenario is that the phorylation during M␾ infection is the following. In WT cells, parasite is restricted to triggering IL-12 through a p38-mediated http://www.jimmunol.org/ pathway involving CREB-dependent C/EBP␤ synthesis. We are MKK3 was the sole p38 specific MKK activated during infection. currently examining the role of these factors during T. gondii However, MKK3 activation kinetics failed to match the p38 phos- infection. phorylation pattern, inasmuch as peak MKK3 phosphorylation lev- Why the parasite would display properties of triggering trans- els were not attained until 60 min postinfection, a time by which duction cascades leading to induction of certain proinflammatory p38 had undergone phosphorylation followed by dephosphoryla- cytokines such as IL-12, while at the same time permanently tion (Fig. 4) (25). In contrast, the kinetics of LPS-induced p38 blocking pathways leading to others such as TNF-␣, is currently a phosphorylation closely paralleled both MKK3 and MKK4 acti- Ϫ/Ϫ ␾ matter for speculation. Regardless, the present study reveals that T. vation. Furthermore, in Toxoplasma-infected MKK3 M , by guest on September 28, 2021 gondii-induced IL-12 requires p38 activation that is dependent there was no detectable activation of MKK3, 4, or 6, yet p38 un- upon parasite-triggered TAB1-dependent p38 autophosphoryla- derwent near normal levels of phosphorylation. tion. As such, the data provide a physiological context for this The pharmacological inhibitor SB203580 functions by occu- unique mode of p38 activation that also suggests a potential target pying the ATP-binding pocket within the p38 kinase cleft, for therapeutic intervention. therefore acting to prevent downstream kinase activity rather than MKK-driven transphosphorylation (50). Thus, the finding Acknowledgments ␣ that SB203580 blocks p38 phosphorylation triggered by T. We thank D. Holowka for insightful discussion and critical review of the gondii is evidence for p38 autophosphorylation. Furthermore, manuscript. pull-down assays revealed a TAB1-p38 association that closely paralleled phosphorylation of the latter (Fig. 6). During LPS Disclosures stimulation, we did not detect an effect of SB203580 on p38 The authors have no financial conflict of interest. activation, nor did we find evidence of TAB1-p38 association. Our results argue that LPS is likely to trigger MKK-driven p38 References ␾ 1. Denkers, E. Y., and R. T. Gazzinelli. 1998. Regulation and function of T cell- phosphorylation in bone marrow-derived M , but that TAB1- mediated immunity during Toxoplasma gondii infection. Clin. Microbiol. Rev. dependent autophosphorylation is a p38 activation mechanism 11:569. triggered by T. gondii infection. The finding that Toxoplasma- 2. Gazzinelli, R. T., S. Hieny, T. Wynn, S. Wolf, and A. Sher. 1993. 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