Transduction of Functionally Contrasting Signals by Two Mycobacterial PPE Proteins Downstream of TLR2 Receptors

This information is current as Atul Udgata, Rahila Qureshi and Sangita Mukhopadhyay of September 27, 2021. J Immunol 2016; 197:1776-1787; Prepublished online 1 August 2016; doi: 10.4049/jimmunol.1501816 http://www.jimmunol.org/content/197/5/1776 Downloaded from

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Transduction of Functionally Contrasting Signals by Two Mycobacterial PPE Proteins Downstream of TLR2 Receptors

Atul Udgata,*,† Rahila Qureshi,* and Sangita Mukhopadhyay*

As pathogen-associated molecular pattern sensors, the TLRs can detect diverse ligands to elicit either proinflammatory or anti- inflammatory responses, but the mechanism that dictates such contrasting immune responses is not well understood. In this work, we demonstrate that proline–proline–glutamic acid (PPE)17 protein of Mycobacterium tuberculosis induces TLR1/2 heterodimerization to elicit proinflammatory-type response, whereas PPE18-induced homodimerization of TLR2 triggers anti-inflammatory type responses. Ligation of TLR1/2 caused an increased recruitment of IL-1R–associated kinase (IRAK)1, MyD88, and protein kinase C (PKC)« to the downstream TLR-signaling complex that translocated PKC« into the nucleus in an IRAK1-dependent manner. PKC«-mediated phosphorylation allowed the nuclear IRAK3 to be exported to the cytoplasm, leading to increased a activation of ERK1/2, stabilization of MAPK phosphatase 1 (MKP-1), and induction of TNF- with concomitant downregulation Downloaded from of p38MAPK. Silencing of TLR1 inhibited PPE17-triggered cytoplasmic export of IRAK3 as well as TNF-a induction, suggesting an important role of TLR1/2 heterodimer in regulating proinflammatory responses via the IRAK3-signaling pathway. In contrast, PPE18-mediated homodimerization of TLR2 caused poorer cytoplasmic export of nuclear IRAK3 and MKP-1 stabilization, resulting in increased p38MAPK activation. Our study hints to a novel mechanism that implicates PKC«–IRAK3–MKP-1 signaling in the regulation of MAPK activity and inflammatory cascades downstream of TLR2 in tuberculosis. The Journal of

Immunology, 2016, 197: 1776–1787. http://www.jimmunol.org/

attern recognition receptors are germline-encoded recep- (9) can bind to TLR2 and interfere with the macrophage innate in- tors that sense infectious agents through molecular sig- flammatory signaling and Th1/Th2 balance (10, 11). P natures known as pathogen-associated molecular patterns TLRs, especially the TLR2, have been reported to be involved in (1, 2). Once activated, they initiate signaling cascades that induce invoking both pro- and anti-inflammatory responses during innate effector immune responses necessary for elimination of the en- activation of macrophages (12, 13). The proinflammatory cytokines countered pathogen. TLRs are one such family of pattern recognition such as TNF-a and IL-12 activate the Th1-type response (14, 15) receptors (3, 4) that are involved in triggering immune responses that is known to induce protective immunity against intracellular against a variety of pathogens, and it comes as no surprise that pathogens such as Mycobacterium tuberculosis (15, 16). Alternately, by guest on September 27, 2021 pathogens have developed unique strategies to hinder TLR-signaling activation of anti-inflammatory cytokines like IL-10 is known cascades (5, 6). For example, Listeria monocytogenes alters its to favor the Th2-type immune environment (15) that helps such peptidoglycan to evade detection by TLR2 (7). Certain bacteria like pathogens to induce a productive infection inside the host (17). Helicobacter pylori produce alternate forms of LPS to prevent de- Thus, the anti- and proinflammatory cytokine balance is crucial to tection by TLR4 (8). Interestingly, the proline–proline–glutamic acid determine the outcome of an intracellular infection. But the exact (PPE) family of proteins that are unique to pathogenic mycobacteria mechanisms by which both of these functionally opposite responses are dictated downstream of the TLR2 are not well understood. In our earlier studies, we reported that the PPE18, a member of the PPE family of proteins of M. tuberculosis upon *Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diag- interaction with TLR2, can induce IL-10 production and anti- nostics, Nampally, Hyderabad 500 001, India; and †Graduate Studies, Manipal University, Manipal, Karnataka 576 104, India inflammatory responses (10), whereas another mycobacterial pro- ORCID: 0000-0002-3633-784X (R.Q.). tein, PPE17, upon interaction with the same receptor, induces TNF-a production and proinflammatory signaling cascades (11). Received for publication August 12, 2015. Accepted for publication June 1, 2016. We hypothesized that such functionally opposite responses of A.U. was supported by a fellowship from the Council of Scientific and Industrial Re- search, Government of India. R.Q. was supported in part by a fellowship from the Indian PPE17 and the PPE18 proteins could be due to their ability to Council of Medical Research, Government of India (67/11/2014-IMM/BMS). The lab- recognize different stretches of the TLR2 ectodomain, resulting oratory of S.M. is supported by Department of Science and Technology, Government of in differential modulation of postreceptor-binding events that lead India Grant SR/SO/HS/0120/2010; Department of Biotechnology, Government of India Grant BT/PR11605/NNT/28/758/2014; and a core grant from the Centre for DNA to activation of pro- and anti-inflammatory responses, respectively Fingerprinting and Diagnostics by the Department of Biotechnology, Government of India. (10, 11). In the current study, we demonstrate that these two PPE Address correspondence and reprint requests to Dr. Sangita Mukhopadhyay, Labo- proteins induce different dimers of TLR2 that lead to a change in ratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics, the protein kinase C (PKC)ε–IL-1R–associated kinase (IRAK3)– Nampally, Hyderabad 500 001, India. E-mail address: [email protected] MAPK phosphatase 1 (MKP-1) signaling axis, which is involved in The online version of this article contains supplemental material. regulation of the TLR2-dependent inflammatory responses in mac- Abbreviations used in this article: CRM1, chromosome region maintenance 1; EGFP, enhanced GFP; IRAK, IL-1R–associated kinase; LMB, leptomycin B; LRR, leucine- rophages. This may have an important impact on our understanding rich repeat; MKP-1, MAPK phosphatase 1; NLS, nuclear localization signal; PKC, of how different ligands can induce TLR2-dependent functionally protein kinase C; PPE, proline–proline–glutamic acid; siRNA, small interfering opposite inflammatory cascades that may be helpful to design ef- RNA; WT, wild type. fective immunomodulators to trigger protective immune responses Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 to control tuberculosis and various intracellular infections. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501816 The Journal of Immunology 1777

Materials and Methods (59-GGAAGAGTTCCCCAGGGACCTCT-39) and a reverse primer (59- 9 a Cloning CCCTTGAAGAGGACCTGGGAGTAG-3 ) specific to TNF- . In brief, RNA was isolated from various groups to synthesize cDNA, as described The pcDNA3-TLR1-YFP was a gift of D. Golenbock (Addgene plasmid by us earlier (10). The amplification condition for MKP-1, TNF-a, and 13014). Myr.PKCepsilon.FLAG and FLAG.PKCepsilon.K/W were gifts of b-actin was as follows: initial denaturation at 95˚C for 5 min, denaturation A. Toker (Addgene plasmid 10797 and plasmid 10796, respectively). RNA at 94˚C for 30 s, annealing at 63˚C (for TNF-a) and 55˚C (for MKP-1) for was extracted from PMA-differentiated THP-1 macrophages using 30 s, extension at 72˚C for 30 s, and final extension for 10 min. After 30 RNAeasy Kit (Qiagen) and was subsequently used for preparing cDNA cycles, the amplified products of MKP-1 (137 bp), TNF-a (380 bp), and using SuperScript Reverse Transcriptase III (Invitrogen, Carlsbad, CA). b-actin (510 bp) were resolved by electrophoresis on 1.2% agarose gels and This cDNA was further used for amplifying TLR2 and IRAK3. For visualized by ethidium bromide staining. amplifying TLR1 and PKCε, pcDNA3-TLR1-YFP (18), Myr-PKCepsilon- FLAG, and FLAG-PKCepsilon-K/W (19) were used as templates, re- Transient transfection spectively. TLR2 was further subcloned into pDsRed using XhoI-SacII HEK293 cells were cotransfected with different combination of plasmids sites. IRAK3 was cloned into pET-Duet using BamHI-SalI sites and then with Lipofectamine 2000 (Invitrogen) and left for 4–6 h. The media were subcloned into pEGFPC3 using BglII-Sal I sites. FLAG-tagged TLR2 was changed, and cells were incubated at 37˚C for 24–36 h and were harvested a gift of C. J. Kirschning (Institute for Medical Microbiology, Immunol- for either immunoprecipitation experiments or confocal microscopy studies. ogy, and Hygiene, Technical University of Munich, Munich, Germany). For expression of PKCε and NLS mutant PKCε, PMA-differentiated THP-1 ε The PKC nuclear localization signal (NLS) mutant was generated by site- macrophages were transfected with the wild-type PKCε (3X-FLAG-PKCε- ε directed mutagenesis, using wild type (WT)-PKC as template. Both the WT) and NLS mutant PKCε (3X-FLAG-PKCε-mutNLS) plasmid constructs ε ε ε WT-PKC and NLS mutant of PKC (mutNLS- PKC ) were further cloned using Lipofectamine LTX (Invitrogen). Briefly, PMA-differentiated THP-1 into BamHI-XhoI site of pcDNA-3X-FLAG vector. macrophages were transfected with the constructs mixed with Lipofect- amine LTX and PLUS reagent in RPMI 1640 (supplemented only with 10% Purification of recombinant proteins Downloaded from FBS) and left for 24 h in a 37˚C incubator, after which they were harvested The PPE17 and PPE18 proteins of M. tuberculosis were expressed and for confocal microscopy studies. purified, as described earlier (10, 11). The recombinant proteins (PPE17 and PPE18) cloned in pRSET A and possessing 63 His tag were purified Small interfering RNA–mediated silencing of genes using TALON (Clonetech, Mountain View, CA) resin, according to man- TLR1-specific small interfering RNA (siRNA) and scrambled siRNA were ufacturer’s protocol. Briefly, primary culture was set up at 37˚C for 12–16 obtained from Sigma-Aldrich. The siRNAs against MKP-1 and IRAK3 h, followed by secondary culture growth and its induction using 1 mM were purchased from Origene (Rockville, MD). pSUPER-PKCε-RNAi was isopropyl b-D-thiogalactoside for 3–4 h at 37˚C. Next, the culture was a gift of A. Toker (Addgene plasmid 10798). The pSUPER backbone http://www.jimmunol.org/ pelleted down and the pellet was resuspended in lysis buffer (13 PBS + 1 vector was a gift of M. Bashyam (Centre for DNA Fingerprinting and mg/ml lysozyme + 1% sodium lauryl sarcosine), sonicated, and centri- Diagnostics, Hyderabad, India). Opti-MEM was purchased from Invi- fuged. Supernatant was collected and loaded on TALON resin column and trogen. Transfection of PMA-differentiated THP-1 macrophages for kept for binding for 30 min at 4˚C, followed by washing with wash buffer siRNA-mediated silencing of TLR1, MKP-1, and IRAK3 was carried out (13 PBS + 20 mM imidazole). Protein was eluted in elution buffer (13 following the protocols as described by the respective manufacturers. Cells PBS + 200 mM imidazole). The purified recombinant protein was dialyzed were harvested 24–36 h after transfection for various experiments. Si- against several changes of PBS. The protein concentration was determined lencing of PKCε in PMA-differentiated THP-1 macrophages was carried using a kit that employs the bicinchoninic acid method of protein esti- out by transient transfection of pSUPER-PKCε-RNAi using Lipofectamine mation (Pierce, Rockford, IL) and was incubated with 10% v/v polymyxin LTX. B-agarose (Sigma-Aldrich, St. Louis, MO) to remove endotoxin, as described earlier (20). The purified proteins had a very low endotoxin content Macrophage activation assay by guest on September 27, 2021 (0.05 EU/ml), as measured by the E-toxate (Limulus amebocyte lysate) kit (Sigma-Aldrich), and were used for all in vitro experiments. PMA-differentiated THP-1 macrophages or human monocyte-derived macrophages were treated with either PPE17 or PPE18 at a concentra- Cell culture tion of 3 mg/ml. Cells were either harvested after 30 min for confocal microscopy or for isolation of total RNA or for preparation of protein The human monocytic cell line THP-1 and human embryonic kidney cell extracts or cultured for 24 h for estimating TNF-a levels in the culture line (HEK293) were obtained from National Centre for Cell Science (Pune, supernatants by enzyme immune assay. In some experiments, cells were India) and maintained in RPMI 1640 medium and DMEM (both supple- pretreated with either leptomycin B (LMB; 20 ng/ml; Sigma-Aldrich) or mented with 10% FBS, L-glutamine, 100 U/ml penicillin, and 100 mg/ml SB203580 (10 mg/ml) or PD98059 (10 mM) or IRAK1/4 inhibitor (2.5 mM) streptomycin; all from Invitrogen, respectively). Human PBMCs obtained or MG-132 (5 mM) (all purchased from Sigma-Aldrich) before incubation from Lonza (Walkersville, MD) were diluted in RPMI 1640 medium with PPE17 and PPE18. (Invitrogen) containing 10% FBS and were differentiated using human GM-CSF (PeproTech, Rocky Hill, NJ). Differentiation of human PBMCs Immunofluorescence and image acquisition was carried out using 400 IU/ml human GM-CSF for 3 d at 37˚C in a 5% Cells were washed twice with 13 PBS and then fixed with 3.7% formal- CO2 humidified incubator. Following this, the cells were replenished with fresh complete RPMI 1640 medium supplemented with GM-CSF, and dehyde for 15 min. The cells were then washed and permeabilized with 0.1% Triton X-100 for 5 min. The cells were next washed and blocked monocyte-derived macrophages were obtained at day 7. THP-1 cells were 3 differentiated to macrophages using 5 ng/ml PMA (Sigma-Aldrich) for with 2% BSA in 1 PBS for 30 min, immunostained with appropriate 12 h, followed by rest for 24 h in complete RPMI 1640 medium. PMA- combinations of primary and secondary Abs, mounted using Vectashield- differentiated THP-1 macrophages were treated with purified recombinant DAPI or Vectashield-PI (Vector Laboratories, Peterborough, U.K.), and PPE17 or PPE18 protein in absence or presence of inhibitors, and cells visualized under an inverted laser-scanning confocal microscope (LSM 700 and LSM 510 META; Carl Zeiss) equipped with a digital microscope were harvested at various time points for Western blotting or immuno- 3 precipitation or immunofluorescence assay using appropriate combinations camera (AxioCam; Carl Zeiss). Images were collected using the 63 oil, of primary and secondary Abs. 1.4 NA, objective lenses and the Zen 2012 acquisition and imaging software (Zen Black 2012, Zen Blue 2012; Carl Zeiss). Image quantification Cytokine assay was performed by using ImageJ software (23). The TNF-a cytokine (BD Pharmingen, San Jose, CA) in the macrophage Immunoprecipitation assay culture supernatants was quantified by two-site sandwich enzyme immune Cells were treated with 1 ml 13 HEPES immunoprecipitation buffer assay following the manufacturer’s protocol, as described earlier (21). (20 mM HEPES [pH 7.5], 1% Nonidet P-40, 2 mM EDTA, 10 mM sodium Standard curves for TNF-a were obtained using the recombinant standard orthovanadate, 10 mM sodium fluoride, and 13 protease inhibitor mixture) protein provided along with the kit. and kept on rotor at 4˚C for 30 min. The samples were next subjected to Semiquantitative RT-PCR for MKP-1 and TNF-a two cycles of freeze thaw and centrifuged. After taking out aliquots of 20 ml from each sample (for input control), the lysates were kept for Semiquantitative RT-PCR was carried out using a forward primer (59- preclearing at 4˚C for 1 h. Following this, 1–2 mg primary Ab was added to CCATCTGCCTTGCTTACCTT-39) and a reverse primer (59- AGCACC- the lysates and kept on rotor at 4˚C for overnight. Next, ∼25 ml pre- TGGGACTCAAACTG-39) specific to MKP-1 (22); and a forward primer equilibrated protein A/G agarose beads (Santa Cruz Biotech, Dallas, TX) 1778 REGULATION OF TLR2 SIGNALING BY PPE PROTEINS were added to each sample and kept on rotor at 4˚C for 3 h. The samples observed in untreated cells. Cells expressing TLR1-EGFP and were washed with immunoprecipitation buffer, boiled with 63 SDS dye, TLR2-DsRed exhibited such dimerization only when cells were and run on a 10% SDS-PAGE for Western blotting using appropriate treated with PPE17, but not with PPE18 (Fig. 1A), whereas cells combinations of primary and secondary Abs. In cases in which the pull- down Ab and the immunoblotting Ab were raised in the same species, expressing TLR2-EGFP and TLR2-DsRed showed diffused yel- Veriblot HRP (Abcam, Cambridge, U.K.) was used. Bound enzyme was low signal when the cells were treated with PPE18, but not with detected by ECL following the manufacturer’s protocol (GE Healthcare), PPE17 (Fig. 1B). This differential dimerization was further con- as described earlier (11). firmed by coimmunoprecipitation experiments, in which FLAG- Nuclear and cytoplasmic extract preparation TLR2 constructs were transiently coexpressed along with either TLR1-EGFP or TLR2-EGFP in HEK293 cells, followed by in- PMA-differentiated THP-1 macrophages were harvested, washed, and cubation with either PPE17 or PPE18 protein. TLR2 was pulled resuspended in cytoplamic extraction buffer (30 mM Tris [pH 7.5], 10 mM magnesium acetate, 1% Nonidet P-40, 1 mM PMSF, 10 mM sodium down with anti-FLAG Ab, and the pulled down complex was orthovanadate, 10 mM sodium fluoride, and 13 protease inhibitor mixture), immunoblotted with either anti-TLR1 Ab or anti-GFP Ab. We followed by intermittent vortexing for 15 min. The extracts were centri- observed an enrichment of the TLR1 band in cells treated with 2 fuged, collected, and stored at 20˚C. For preparing nuclear extracts, cells PPE17, indicating that the protein recruited heterodimer of TLR1 were washed with cytoplasmic buffer, and the pellets were resuspended with nuclear extraction buffer (10 mM HEPES [pH 8], 25% glycerol, and TLR2 (Fig. 1C, 1D). However, enrichment of GFP band was 10 mm magnesium chloride, 1 mM EDTA, 1 mM PMSF, 400 mM sodium observed in cells treated with PPE18, thus indicating recruitment chloride, 10 mM sodium orthovanadate, 10 mM sodium fluoride, and 13 of homodimeric TLR2 by PPE18 (Fig. 1E, 1F). These results protease inhibitor mixture), followed by intermittent vortexing for 1 h at indicate that contrastive signaling events triggered by PPE17 and

4˚C. The nuclear fractions were collected after centrifugation and stored. PPE18 are due to recruitment of TLR1/2 heterodimer by PPE17 Downloaded from Reagents and Abs and TLR2 homodimer by PPE18. Rabbit anti-IRAK3 Ab (ab8116) was purchased from Abcam; rabbit anti- PPE17 causes translocation of IRAK3 into the cytosol PKCε Ab (C-15), rabbit anti–MKP-1 Ab (C-19), goat anti-Lamin B (C-20), b We had shown earlier that PPE17 predominantly induced the and rabbit anti– -tubulin Ab (H-235) were purchased from Santa Cruz; k a rabbit anti-ERK1/2 Ab (9102), mouse anti–phospho-ERK1/2 Ab (9106), NF- B and the proinflammatory cytokine TNF- (11), whereas k rabbit anti-TLR1 Ab (2209), rabbit anti-MyD88 Ab (D80F5), and rabbit PPE18 inhibited nuclear NF- B signaling and activated induction http://www.jimmunol.org/ anti-IRAK1 Ab (D51G7) were purchased from Cell Signaling Technology of the anti-inflammatory cytokine IL-10 in macrophages (10, 11, (Danvers, MA); mouse anti-p38MAPK Ab (612169) and mouse anti– 24). We therefore looked at molecules involved in regulating this phospho-p38MAPK Ab (612288) were purchased from BD Biosciences (San Jose, CA); and rabbit anti-GFP Ab (G1544), anti-mouse Alexa Fluor cytokine signaling downstream of TLR2. IRAK3, a member of the 594 Ab, and mouse anti-FLAG Ab (F1804) were purchased from Sigma- IRAK family that lacks the critical aspartate residue (25), is known Aldrich. Anti-rabbit Alexa Fluor 488 Ab was obtained from Invitrogen. to regulate TLR signaling (26, 27). Pam3CysK4, a synthetic lipopeptide, is an agonist for TLR1/2 and triggers proinflammatory Mycobacterium smegmatis culture and transformation signaling cascades and TNF-a production (28). Upon Pam3CysK4 M. smegmatis mc2155 was grown in 7H9 media with albumin-dextrose- stimulation in bone marrow–derived macrophages, IRAK3 is shown catalase supplement (HIMEDIA, Mumbai, India), 0.05% Tween 80, to be present predominantly in the cytosol, whereas, in untreated by guest on September 27, 2021 and 0.2% glycerol till log phase, as described earlier (11). To transform cells, it remains diffused throughout the cells (27). This study in- M. smegmatis with pVV16-PPE constructs, electro-competent cells were prepared. Briefly, log-phase M. smegmatis were pelleted down and washed dicates that Pam3CysK4 triggers translocation of IRAK3 from the with ice-cold 10% glycerol, and aliquots of 100 ml were prepared in nucleus to the cytosol. Because Pam3CysK4 binds to a heterodimer microcentrifuge tubes and stored at 270˚C. Cells were transformed by of TLR1/2 (29), we questioned whether PPE17 that stabilized a electroporation (11) with Gene Pulser (Bio-Rad, Hercules, CA), and trans- TLR1/2 heterodimer also targeted the IRAK3 signaling in macro- formants were grown on 7H9 Agar plates containing 50 mg/ml hygromycin B (Invitrogen) and 50 mg/ml kanamycin (Amresco, Solon, OH). phages to induce a proinflammatory response in macrophages. PMA-differentiated THP-1 macrophages were therefore treated with PPE17 or PPE18 or Pam3CysK4; cells were fixed and per- Results meabilized; and IRAK3 distribution was visualized by confocal PPE17 and PPE18 cause differential dimerization of TLR2 microscopy. It was observed that, in untreated and PPE18-treated In our earlier studies, we had shown that the recombinant PPE17 macrophages, IRAK3 had localized throughout the cell and was and PPE18 proteins bind to TLR2 at different regions and elicit present both in the cytoplasm and in the nucleus (Fig. 2A). How- different signaling cascades (10, 11, 24). Although PPE17 was ever, in macrophages treated with PPE17 or Pam3CysK4,IRAK3 found to bind to the leucine-rich repeat (LRR) 16–20 and induced had relocalized predominantly to the cytoplasm (Fig. 2A). Also, a proinflammatory-type response, PPE18 interacted with the LRR when human PBMC-derived macrophages were treated with PPE17 11–15 and triggered an anti-inflammatory type response. We hy- or PPE18, IRAK3 was found to be exported from the nucleus and pothesized that these opposing responses could either be a result distributed in the cytoplasm of PPE17-treated macrophages (Fig. 2B). of a conformational change in TLR2 or because of differential However, in control and PPE18-treated macrophages, IRAK3 was dimerization of TLR2. To explore this, we transiently coexpressed located both in the nucleus and in the cytoplasm (Fig. 2B). We further TLR1-enhanced GFP (EGFP) or TLR2-EGFP and TLR2-DsRed evaluated IRAK3 localization using M. smegmatis bacilli expressing in HEK293 cells and treated the cells with either recombinantly PPE17 (pVV16-PPE17 M. smegmatis)orPPE18(pVV16-PPE18 purified PPE17 or PPE18 protein and checked under confocal M. smegmatis). As expected, export of nuclear IRAK3 into the cy- microscopy. If dimerization did occur, it would be scored by a toplasm was more prominent in THP-1 macrophages infected with yellow signal caused by colocalization of EGFP and DsRed pro- PPE17-expressing M. smegmatis as compared with PPE18-expressing teins. We observed that, in untreated cells, there were yellow M. smegmatis (Supplemental Fig. 1). punctate-like structures, which indicated that some amounts of We next used the online database NetNES 1.1 to look for the TLRs were in close vicinity and could possibly form a complex in presence of nuclear export signal in IRAK3, and a nuclear export the absence of any ligand. However, upon addition of ligands, signal was predicted (27, 30) (Fig. 2C). LMB is known to be a potent dimerization of TLR2 was evident from the spread of yellow signal inhibitor of chromosome region maintenance 1 (CRM1) nuclear on the cell surface in comparison with the punctate structures export (31). We pretreated macrophages with LMB followed by The Journal of Immunology 1779 Downloaded from http://www.jimmunol.org/

FIGURE 1. Differential dimerization of TLR2 by PPE17 and PPE18 proteins. (A and B) TLR1-EGFP and TLR2-DsRed (A) or TLR2-EGFP and TLR2- DsRed (B) transiently coexpressed in HEK293 cells were either left untreated (Medium) or treated with recombinantly puriﬁed PPE17 or PPE18 protein at 10 mg/ml concentration for 30 min, and cells were observed under a confocal microscope. Results are representative of three different experiments. Scale

bars, 5mm. (C and D) TLR1-EGFP along with FLAG-TLR2 were transiently coexpressed in HEK293 cells and were either left untreated (Medium) or by guest on September 27, 2021 treated with PPE17 or PPE18 protein (10 mg/ml for 30 min). Cells were lysed, and TLR2 was pulled down using anti-FLAG Ab and immunoblotted using anti-TLR1 Ab (C). About 10% of the lysate was run as input control. Densitometric analyses of the Western blots were carried out using ImageJ software using TLR1-EGFP as loading control (D). Results are representative of mean 6 SD of three different experiments. (E and F) TLR2-EGFP with FLAG- TLR2 were transiently coexpressed in HEK293 cells and were either left untreated (Medium) or treated with PPE17 or PPE18 protein (10 mg/ml for 30 min). Cells were lysed, and TLR2 was pulled down using anti-FLAG Ab and immunoblotted using anti-GFP Ab (E). About 10% of the lysate was run as input control. Densitometric analyses were carried out using ImageJ software, and TLR2-EGFP was used as loading control (F). Results are representative of mean 6 SD of three different experiments. treatment with PPE17 or PPE18 protein to test whether export of K treatment reduced the expression of TNF-a in response to nuclear IRAK3 in response to PPE17 is CRM1 dependent. The PPE17. Similarly, proteinase K treatment inhibited the PPE18- confocal microscopy data indicate that the export of IRAK3 from induced expression of IL-10 cytokine. These studies indicate the nucleus to the cytosol in PPE17-treated macrophages is indeed that the observed effects are due to PPE17 and PPE18 proteins. CRM1 dependent (Fig. 2D). As expected (27), IRAK3 export was We also silenced the TLR4 expression in THP-1 macrophages found to be LMB sensitive in macrophages treated with Pam3CysK4 using a TLR4-specific siRNA (Supplemental Fig. 2E) and examined (Fig. 2D). These results suggest that redistribution of IRAK3 by the levels of TNF-a induced by PPE17 and IL-10 by PPE18 pro- PPE17 requires nuclear export machinery. tein, respectively. We observed that induction of TNF-a by PPE17 To rule out the possibility that the PPE17-mediated nuclear was not affected by silencing of TLR4 expression in THP-1 mac- export of IRAK3 in THP-1 macrophages was due to endotoxin rophages (Supplemental Fig. 2F). Similarly, PPE18-mediated in- contamination in the protein preparation, we treated the recom- duction of IL-10 in THP-1 macrophages was unaffected when binantly purified PPE17 or PPE18 protein with proteinase K, a TLR4 expression was knocked down using TLR4-specific siRNA serine protease known to digest and denature proteins (32). Pro- (Supplemental Fig. 2G). All these experiments together confirm teinase K–treated PPE17 failed to significantly translocate IRAK3 that the observed signaling events and the effector responses trig- from the nucleus to the cytoplasm (Supplemental Fig. 2A, 2B), gered in THP-1 macrophages are specific to the PPE proteins, but indicating that observed nuclear export of IRAK3 is indeed due to are not due to contamination of PPE proteins (PPE17 and PPE18) PPE17 protein, but not due to contaminating LPS. Because PPE17 by LPS, which binds to TLR4 for its signaling. has been shown to activate TNF-a production in macrophages (11) and PPE18 is found to trigger IL-10 induction (10), we also Inhibition of IRAK3 export reverses the proinflammatory examined the level of TNF-a and IL-10 cytokines in macrophages signaling triggered by PPE17 in macrophages incubated with proteinase K–treated PPE17 and PPE18 protein, ERK1/2 and p38MAPK have been implicated in regulation of respectively (Supplemental Fig. 2C, 2D). We found that proteinase cytokine production in response to TLR2-triggered signaling, with 1780 REGULATION OF TLR2 SIGNALING BY PPE PROTEINS

FIGURE 2. PPE17 causes relocalization of IRAK3 into the cytosol. (A and B) THP-1 macrophages (A) or human PBMC-derived macrophages (B) were either left untreated (Medium) or treated with either Pam3CysK4 or PPE17 or PPE18 protein at 3 mg/ml concentration for 30 min. Cells were ﬁxed, permeabilized, and stained with anti-IRAK3 Ab, followed by anti- rabbit Alexa Fluor 488 Ab to visualize IRAK3 distribution under confocal microscope. Nucleus was visualized by staining with either propidium iodide (PI) (A)orDAPI(B). (C)Presenceof nuclear export signal in IRAK3 sequence was checked (highlighted in red) using NetNES 1.1 Server. (D) THP-1 macrophages were pretreated with 20 ng/ml LMB for 3 h and then either left untreated or treated with either Pam3CysK4 or PPE17 or PPE18 protein at 3 mg/ml concentration for 30 min. Cells were ﬁxed and permeabilized,

and IRAK3 distribution was visualized under Downloaded from confocal microscope by staining the cells with anti-IRAK3 Ab, followed by anti-rabbit Alexa Fluor 488 Ab. Nucleus was visualized by staining with propidium iodide. Results are representative of three different experiments. Scale bars, 5mm. http://www.jimmunol.org/

ERK1/2 being responsible for TNF-a induction and p38MAPK for cells and found that MKP-1 level was higher as compared with IL-10 production (12, 20, 33–35). We had observed earlier that that of untreated or PPE18-treated macrophages (Fig. 4A). Inter- PPE18 strongly activated p38MAPK (but not ERK1/2) that was estingly, the mRNA levels of MKP-1 did not differ significantly in necessary for the activation of IL-10 (10). Because PPE17 was all of the three groups examined (untreated and PPE17- and found to activate predominantly the proinflammatory cytokine like PPE18-treated macrophages) (Fig. 4B); thus, the observed re- TNF-a (11), we expected higher activation of ERK1/2 in PPE17- duction in the protein levels of MKP-1 could be attributed to treated macrophages as compared with PPE18-treated macro- decreased stability of the protein in untreated and PPE18-treated phages. Our data indicated that indeed the level of p38MAPK macrophages. MKP-1 is known to be a labile protein and un- by guest on September 27, 2021 phosphorylation was lower, but ERK1/2 phosphorylation level dergoes rapid turnover through proteasome-mediated degradation was higher in PPE17-treated macrophages when compared with (27, 40). We therefore next pretreated cells with MG132, a pro- PPE18-treated macrophages (Fig. 3A). Pretreatment of THP-1 teasome inhibitor, followed by incubation with medium alone macrophages with SB203580, an inhibitor of p38MAPK activ- or PPE18 protein. MG132 was found to increase the levels of ity, resulted in increased phosphorylation of ERK1/2 in PPE18- MKP-1 in both medium-treated and PPE18-treated macrophages treated macrophages, indicating that p38MAPK pathway probably (Fig. 4C). We then examined whether IRAK3-export mechanism negatively regulated ERK1/2 activation in PPE18-treated macro- was essential for MKP-1 stability in PPE17-treated macrophages. phages (Fig. 3B). We next examined whether ERK1/2 activity was It was observed that the levels of MKP-1 decreased when LMB required for TNF-a induction by PPE17. When cells were treated was used to inhibit export of nuclear IRAK3 in these macrophages with PD98059, an inhibitor of ERK1/2 activity, TNF-a production (Fig. 4D). To confirm whether presence of cytosolic IRAK3 is in PPE17-treated macrophages was found to be inhibited. This important for stabilization of MKP-1, we next silenced IRAK3 result confirmed that TNF-a induction by PPE17 was dependent expression in THP-1 macrophages using IRAK3-specific siRNA upon ERK1/2 activity (Fig. 3C). In the previous section, we (Fig. 4E), and, following treatment with PPE17, MKP-1 levels demonstrated that export of nuclear IRAK3 into the cytosol in were examined. It was observed that the levels of MKP-1 were PPE17-treated macrophages was inhibited by LMB. So we ex- poorer in THP-1 macrophages transfected with IRAK3-specific amined whether blocking export of nuclear IRAK3 to cytoplasm siRNA as compared with the MKP-1 levels in macrophages had any effect on ERK1/2 phosphorylation and TNF-a induc- transfected with scrambled siRNA (Fig. 4E). These results to- tion in PPE17-treated macrophages. We found that LMB could gether indicated that the export of IRAK3 to the cytoplasm in inhibit phosphorylation of ERK1/2 and induction of TNF-a (Fig. PPE17-treated macrophages was necessary to maintain MKP-1 3D, 3E) and enhance the level of phosphorylated p38MAPK in stability, resulting in reduced phosphorylation of p38MAPK these cells (Fig. 3F) and thereby be able to mimic the PPE18 with simultaneous upregulation of phospho-ERK1/2 and TNF-a phenotype. These data indicate that the export of nuclear IRAK3 levels. to the cytoplasm is necessary for inhibition of p38MAPK activa- To prove a role of MKP-1 in dictating PPE17-induced MAPK tion with simultaneous activation of ERK1/2 and TNF-a cytokine signaling cascades, we next silenced MKP-1 expression in THP-1 in PPE17-treated macrophages. macrophages using a MKP-1-specific siRNA (Fig. 4F) and ex- The MKP-1 is known to dephosphorylate MAPKs (36). Evi- amined MAPK activation following PPE17 treatment. It was ob- dence suggests that MKP-1 can suppress p38MAPK activation, served that the cells that received the MKP-1–specific siRNA had but does not affect ERK1/2 or JNK activation (28, 37–39). Be- reduced ERK1/2, but increased p38MAPK phosphorylation cause we observed a reduction in p38MAPK activity in PPE17- (Fig. 4G), resulting in significant reduction in TNF-a production treated macrophages, we examined the levels of MKP-1 in these as compared with the cells that received the scrambled siRNA The Journal of Immunology 1781

FIGURE 3. LMB treatment reverses the PPE17- induced proinﬂammatory response environment in macrophages. (A) THP-1 macrophages treated with 3 mg/ml PPE17 or PPE18 protein (30 min) were lysed, and the levels of phosphorylated p38MAPK (p-p38MAPK) and total p38MAPK as well as phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 levels were measured by Western blotting. (B–D) THP-1 macrophages were pretreated with either 10 mg/ml SB203580 for 4 h (B)or10mM PD98059 for 4 h (C) or 20 ng/ml LMB for 3 h (D) and then either left untreated (Medium) or treated for 30 min with 3 mg/ml PPE18 (B) or PPE17 (C and D). Cells were either lysed to measure the levels of phosphorylated and total ERK1/2 levels by Western blotting (B and D) or processed for RNA extraction and subsequent cDNA preparation for measuring TNF-a and b-actin mRNA levels by semiquantitative RT-PCR (C). Results are representative of three

different experiments. (E) THP-1 macrophages pre- Downloaded from treated with LMB were incubated with 3 mg/ml either PPE17 or PPE18 protein, and, after 24 h, TNF-a cytokine levels were quantiﬁed in different culture supernatants by enzyme immune assay. Data are representative of mean 6 SD of three different experiments. (F) THP-1 macrophages were treated with PPE17 (3 mg/ml for 30 min) in presence of http://www.jimmunol.org/ 20 ng/ml LMB. Cells were lysed for immunoblotting to measure the levels of phosphorylated and total p38MAPK. Results are representative of three different experiments.

(Fig. 4H). This study further confirms our previous observation nucleus, the NLS mutant showed reduced nuclear translocation that MKP-1 has a pivotal role in influencing the MAPK pathway (Fig. 5A). Thus, we speculated that PKCε translocates to the and TNF-a induction downstream of PPE17-induced signaling nucleus to phosphorylate nuclear IRAK3. We next coexpressed by guest on September 27, 2021 events. Thus, our study hints to an existence of a link between GFP-tagged IRAK3 along with WT-PKCε or Mut-PKCε [where IRAK3, MKP-1, and MAPK signaling cascades downstream of the lysine residue in its substrate binding domain was replaced by TLR2 that plays an important role in dictating the pro- and anti- a tryptophan, which makes it unable to bind and phosphorylate its inflammatory cytokine responses. substrates (19)] in HEK293 cells and found that, although both the WT-PKCε and Mut-PKCε were localized to the nucleus (Fig. 5B), « IRAK3 is a target of PKC nuclear IRAK3 could translocate to the cytoplasm only in cells Because phosphorylation is often implicated in shuttling of pro- overexpressing WT-PKCε, but not Mut-PKCε (Fig. 5C). This in- teins between various compartments of the cell (41), we speculated dicated that the kinase activity of PKCε was probably essential for that IRAK3 would probably be phosphorylated during its trans- the nuclear export of IRAK3 to the cytoplasm. We next tested location from the nucleus to the cytosol. In silico analyses of the whether IRAK3 was truly phosphorylated by WT-PKCε in this polypeptide sequence of IRAK3, using NetPhosK (42) and GPS experimental setup. When IRAK3 was pulled down using anti- (43), revealed that IRAK3 contained four possible phosphoryla- GFP Ab and subsequently probed with anti-phosphoserine Ab, we tion sites for PKC isoform, PKCε. PKCε is a member of the PKC observed a prominent phosphoserine signal in IRAK3 that was family of kinases that is shown to have diverse roles in the cellular coexpressed with WT-PKCε (Fig. 5D). To identify the IRAK3 physiology (44) and is recruited to the TLR signaling pathways phosphorylation site, we expressed His-tagged IRAK3 either via the MyD88 adaptor protein (45). We, therefore, questioned alone or along with WT-PKCε in HEK293 cells using transient whether PKCε had a direct role in the phosphorylation and export transfection, and, after 36 h, IRAK3 was pulled down using Ni- of IRAK3 from the nucleus to the cytoplasm. To facilitate phos- NTA beads, and the samples were run on a SDS-PAGE and the phorylation and nuclear export of IRAK3, PKCε should be lo- IRAK3 band was cut and sent for mass spectrometry and tandem calized to the nucleus. Therefore, we looked for the presence of mass spectrometry analysis to TAPLIN Mass Spectrometry Fa- NLS in PKCε using NucPred tool (46) and found the presence cility (Harvard). Upon analysis, it was observed that IRAK3 was of one putative NLS (319RRKK322) motif. To prove the fact that phosphorylated at Ser110 site in the sample in which IRAK3 was the nuclear translocation of PKCε was truly dependent on the expressed along with WT-PKCε (Supplemental Fig. 3). Thus, NLS, we next mutated this putative NLS 319RRKK322 motif to PKCε appears to function as an important point of signal regu- 319GGAA322 and then examined the localization of the PKCε in lation, facilitating phosphorylation and translocation of IRAK3 THP-1 macrophages. Briefly, THP-1 macrophages were transfected from the nucleus to the cytosol, which is important for activation with either 3X-FLAG-WT-PKCε or 3X-FLAG-mutNLS-PKCε of ERK1/2, stabilization of MKP-1, with concomitant downreg- followed by treatment with PPE17 (3 mg/ml), and localization of ulation of p-p38MAPK. Thus, MAPK activity in PPE17-treated FLAG-tagged PKCε was analyzed by confocal microscopy. We macrophages is probably influenced upstream by PKCε. To prove found that, although WT-PKCε was able to translocate to the this, we have now silenced PKCε expression using a short hairpin 1782 REGULATION OF TLR2 SIGNALING BY PPE PROTEINS Downloaded from

a A B m FIGURE 4. IRAK3 influences the MAPK pathway and induction of TNF- through MKP-1. ( and ) THP-1 macrophages were treated with 3 g/ml http://www.jimmunol.org/ PPE17 or PPE18 protein for 30 min, and cells were lysed for immunoblotting to measure the levels of MKP-1 and b-actin (A) or used to isolate RNA to measure MKP-1 and b-actin levels by semiquantitative RT-PCR (B). Results are representative of three different experiments. (C) THP-1 macrophages were treated with PPE18 (3 mg/ml for 30 min) in presence of 5 mM MG-132. Cells were then lysed, and presence of MKP-1 in the lysates was detected by Western blotting using anti–MKP-1 Ab. Equal loading of samples was confirmed by probing the same blots with anti–b-actin Ab. Results shown are representative of three different experiments. (D) THP-1 macrophages were treated with PPE17 (3 mg/ml for 30 min) in presence of 20 ng/ml LMB. Cells were then lysed, and presence of MKP-1 in the lysates was detected by Western blotting using anti–MKP-1 Ab. Equal loading of samples was confirmed by probing the same blots with anti–b-actin Ab. Results shown are representative of three different experiments. (E) THP-1 macrophages were transfected with either IRAK3-specific siRNA or scrambled siRNA as control. After 24 h of transfection, cells were treated with 3 mg/ml PPE17 for 30 min, lysed, and immunoblotted with anti-IRAK3 Ab and anti–MKP-1 Ab, respectively. Results shown are representative of three different experiments. (F–H) THP-1 macrophages were transfected with either MKP-1–specific siRNA or scrambled siRNA, and, after 24 h of transfection, cells were treated with 3 mg/ml by guest on September 27, 2021 PPE17 for 30 min. Post-PPE17 treatment, cells were either lysed and immunoblotted to check the levels of either MKP-1 (F) or phosphorylated p38MAPK (p-p38MAPK) and total p38MAPK as well as phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 (G) or processed for RNA extraction and subsequent cDNA preparation for measuring TNF-a and b-actin mRNA levels by semiquantitative RT-PCR (H). Results shown are representative of three different experiments.

RNA construct (pSUPER-PKCε-RNAi) in THP-1 macrophages of MyD88-associated PKCε in cells treated with PPE17 or PPE18. (Fig. 6A). When these cells were treated with PPE17, it was ob- The PKCε recruitment was found to be higher in PPE17-treated served that knock down of PKCε led to a decrease in export of macrophages as compared with PPE18-treated macrophages (Fig. IRAK3 from the nucleus to the cytosol (Supplemental Fig. 4) with 7A). As there were no signiﬁcant changes in the protein levels of a concomitant reduction in p-ERK1/2 but increased p-p38MAPK IRAK1, MyD88, and PKCε in all of the three groups tested (Fig. 7A), levels as compared with the control cells that received only the it appears that PPE17 is more proﬁcient in recruiting these adaptor backbone vector (Fig. 6B). These results indicate that PKCε proteins downstream of TLR1/2 complex. plays a crucial role in regulating nuclear export of IRAK3 and We next investigated the compartmental localization of PKCε MAPK activity downstream of TLR2 in PPE17-treated THP-1 in response to PPE17 and PPE18 treatment. PMA-differentiated macrophages THP-1 macrophages were treated with either PPE17 or PPE18 for 30 min, following which cells were immunostained with anti- « IRAK3 translocation is dependent on the IRAK1–PKC axis PKCε Ab. Upon examination by confocal microscopy, we found The TLR2 signaling pathway is known to recruit adaptor proteins an increased localization of PKCε inside the nucleus in PPE17- MyD88 and IRAK1, which are responsible for transducing stimuli treated macrophages as compared with untreated macrophages or downstream of TLR2 once the receptor is engaged by its cognate PPE18-treated macrophages (Fig. 7B, 7C). Similar observation ligand (47). Because PPE17 and PPE18 induced two different was made in primary human macrophages (Fig. 7B, 7D). Because types of signaling cascades downstream of TLR2, we next ex- we observed recruitment of PKCε to the IRAK1–MyD88 com- amined whether there was any change in the recruitment of plex (Fig. 7A) and IRAK1 has been shown to interact with PKCε MyD88 and IRAK1. Extracts prepared from PMA-differentiated (48), we therefore investigated whether translocation of PKCε THP-1 macrophages treated with PPE17 or PPE18 protein were to the nucleus was dependent on IRAK1 activity. Therefore, used to pull down MyD88–IRAK1 complex with anti-IRAK1 Ab. PMA-differentiated THP-1 macrophages and primary human Immunoblotting of the complex with anti-MyD88 Ab indicated an macrophages were either left untreated or pretreated with N-(2- increased recruitment of MyD88 in cells treated with PPE17 as morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole, an compared with the cells treated with PPE18 (Fig. 7A). Next, we inhibitor of IRAK1/4 activity, as described by various groups (49, probed the same extracts with anti-PKCε Ab to compare the level 50), followed by treatment with either PPE17 or PPE18 protein, The Journal of Immunology 1783 Downloaded from

FIGURE 5. PKC-mediated phosphorylation is required for transport of IRAK3 from the nucleus into the cytosol. (A) THP-1 macrophages were transfected with either 3X-FLAG-WT-PKCε or 3X-FLAG-mutNLS-PKCε and were treated with 3 mg/ml PPE17 protein for 30 min. Cells were fixed, permeabilized, and stained with anti-FLAG Ab, followed by anti-mouse Alexa Fluor 594 Ab. Data shown are representative of three independent ex- http://www.jimmunol.org/ periments. (B) HEK293 cells were transiently cotransfected with either pcDNA-6xHis-WT-PKCε or pcDNA-6xHis-Mut-PKCε along with pEGFPC3- IRAK3. After 24 h, cells were harvested to check the level of PKCε in the cytoplasmic and nuclear extracts using anti-PKCε Ab, and equal sample loading was confirmed by probing the blots with anti–b-tubulin Ab for cytoplasmic extract and anti-Lamin B Ab for nuclear extracts. Results shown are representative of three independent experiments. (C and D) HEK293 cells transiently cotransfected with either pcDNA-6xHis-PKCε-WT or pcDNA-6xHis- PKCε-Mut along with pEGFPC3-IRAK3 were either analyzed for IRAK3 localization by confocal microscopy (C) or harvested to prepare lysate to check the phosphorylated IRAK3 levels using anti-GFP Ab for immunoprecipitation (IP) and anti-phosphoserine (anti-pSerine) Ab for immunoblotting (IB) (D). About 10% of the lysates were loaded as input controls. Results shown are representative of three independent experiments. Scale bars, 5mm. and PKCε distribution was visualized by confocal microscopy. It alone exhibited cytoplasmic export of nuclear IRAK3 upon PPE17 by guest on September 27, 2021 was observed that the IRAK 1/4 inhibitor reduced nuclear trans- treatment (Fig. 9B, 9C). This further substantiates our claim that location of PKCε in PPE17-treated macrophages (Fig. 7B–D). PPE17 does signal through the TLR1/2 heterodimer, which is These results suggest that migration of PKCε to the nucleus upon responsible for the export of IRAK3 from the nucleus to the cyto- PPE17 treatment is dependent upon the activity of IRAK1. Be- sol. Because PPE17 triggers TNF-a induction, next we examined cause export of IRAK3 from the nucleus to the cytosol was found whether the TLR1/2 signaling was required for the induction of to be dependent on PKCε activity (Fig. 5) and nuclear import of TNF-a in PPE17-treated macrophages. Therefore, PMA-differentiated PKCε was dependent on IRAK1 activity (Fig. 7), we next inves- THP-1 macrophages were transfected with either TLR1-siRNA or tigated whether the inhibition of IRAK1 activity had any effect on IRAK3 translocation. PMA-differentiated THP-1 macrophages and primary human macrophages were treated with PPE17 or PPE18 in the absence or presence of IRAK1/4 activity inhibitor, and IRAK3 distribution was assessed by confocal microscopy. The IRAK1/4 inhibitor reduced translocation of IRAK3 from nucleus to cytosol in PPE17-treated macrophages (Fig. 8). As expected, IRAK1/4 inhibitor did not strongly affect translocation of IRAK3 in untreated and PPE18-treated macrophages (Fig. 8). Silencing of TLR1 in PPE17-treated macrophages blocked IRAK3 translocation from the nucleus into the cytosol and suppressed TNF-a production In the previous section, we provided evidence that PPE17-triggered TLR1/2 heterodimer formation and the downstream adaptor complex-associated signaling cascades mediated translocation of ε IRAK3 and subsequent induction of proinflammatory responses. FIGURE 6. PKC dictates MAPK activation in PPE17-treated macrophages. (A and B) PMA-differentiated THP-1 macrophages were transfected We next examined whether downstream events of TLR1 have any with either pSUPER or pSUPER-PKCε-RNAi, and, at 24 h posttransfection, role on export of nuclear IRAK3 in PPE17-treated macrophages by cells were either lysed and immunoblotted with anti-PKCε Ab (A)ortreated silencing TLR1 expression using a TLR1-specific siRNA (Fig. 9A). with 3 mg/ml PPE17 for 30 min and lysed and immunoblotted to check the The confocal analysis indicated that export of IRAK3 from the levels of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 or phos- nucleus to cytosol was stalled in cells transfected with TLR1 phorylated p38MAPK (p-p38MAPK) and total p38MAPK (B). Results siRNA, whereas cells that received scrambled siRNA or medium shown are representative of three independent experiments. 1784 REGULATION OF TLR2 SIGNALING BY PPE PROTEINS Downloaded from http://www.jimmunol.org/

FIGURE 7. Nuclear localization of PKCε in PPE17-treated macrophages is dependent on IRAK1 activity. (A) THP-1 macrophages were treated with

3 mg/ml PPE17 or PPE18 protein for 30 min and then lysed. Equal amounts of cell extracts were incubated with anti-IRAK1 Ab bound to protein A/G by guest on September 27, 2021 agarose beads. Immunoprecipitated complexes were separated on a 10% SDS-PAGE and transferred to a nitrocellulose membrane, which was probed with either anti-MyD88 Ab or anti-PKCε Ab. About 10% of the lysates were loaded as input controls. Results shown are representative of three independent experiments. (B) THP-1 macrophages as well as human PBMC-derived macrophages were either left untreated or pretreated with 2.5 mM IRAK1/4 inhibitor for 3 h, and then incubated with 3 mg/ml PPE17 or PPE18 protein. After 30 min, cells were fixed, permeabilized, and stained with anti-IRAK3 Ab, followed by anti-rabbit Alexa Fluor 488 Ab. Scale bars, 5mm. (C and D) Image quantification of nuclear import of PKCε in THP-1 macrophages (n = 23) (C) or human PBMC-derived macrophages (n = 30) (D) with or without IRAK1/4 inhibitor was carried out using ImageJ software. Data shown are representative of three independent experiments. scrambled siRNA or medium alone, followed by treatment with In our earlier studies, we have reported that two members of PPE17, and mRNA was isolated for estimating TNF-a expression the PPE family of proteins of M. tuberculosis, PPE17 and PPE18, levels. We observed that silencing of TLR1 resulted in downreg- induced antagonistic inflammatory responses upon interaction ulation of PPE17-induced TNF-a production (Fig. 9D). Therefore, with TLR2 (10, 11, 24). The reasons for such contrasting immune it appears that PPE17 requires transduction of signals through the responses emanating from the same TLR2 when engaged to two TLR1/2 heterodimer to induce TNF-a production via IRAK3- different proteins are unique. One plausible explanation is that mediated signaling pathway. these two proteins induce different dimerization of TLR2 with itself or other TLR members. TLR2 is known to form dimers with Discussion TLR1 and TLR6 to recognize triacylated lipopeptides and TLRs belong to the family of pattern recognition receptors that diacylated lipopeptides, respectively (2). Similarly, we observed detect molecular signatures in the pathogen and alert the host that PPE17 induced TLR1/2 heterodimerization, whereas PPE18 immune system to mount an appropriate response (3, 4). The caused homodimerization of TLR2. We also observed some extent signaling cascades triggered once the TLR2 has been engaged by of heterodimerization of TLR1/2 in the presence of PPE18, its corresponding ligand are mostly proinflammatory in nature and homodimerization of TLR2 in the presence of PPE17, as well as are well documented (51). However, TLR2 has also been shown to both forms of dimerization in the absence of any ligand. This could initiate anti-inflammatory cascades (13, 52), but the mechanisms be a result of dynamic equilibrium between the individual recep- by which the anti- and proinflammatory signaling cascades are tors. In fact, TLR1 and TLR2 are known to interact in the absence regulated downstream of TLR2 remain poorly understood. As of any ligand (18). To the best of our knowledge, this study dem- proinflammatory cascades are important in providing protection onstrates for the first time that TLR2 can form a homodimer or a against the intracellular pathogens, they are often manipulated by heterodimer with TLR1 when engaged by the respective mycobac- the pathogens to escape the host immune system to favor its terial ligands, leading to contrasting anti-inflammatory and proin- survival inside the host (53). flammatory cytokine signaling. This opens up a new mechanism by The Journal of Immunology 1785

FIGURE 8. IRAK1 activity is required for the export of nuclear IRAK3 to the cytoplasm of PPE17-treated macrophages. (A) THP-1 macrophages and human PBMC-derived macrophages were either left untreated or pretreated with IRAK1/4 activity inhibitor (2.5 mM for 3 h), and then incubated with Downloaded from 3 mg/ml PPE17 or PPE18 protein. After 30 min, cells were fixed, permeabilized, and stained with anti-PKCε Ab, followed by anti-rabbit Alexa Fluor 488 Ab. Scale bars, 5mm. (B and C) Image quantification of nuclear export of IRAK3 in THP-1 macrophages (B) or human PBMC-derived macrophages (C) with or without IRAK1/4 inhibitor was carried out using ImageJ software. On an average, 30 cells per experiment were used for quantification of nuclear export of IRAK3 in both THP-1 macrophages (B) as well as human PBMC-derived macrophages (C). Data shown are representative of three independent experiments. http://www.jimmunol.org/ which TLRs can increase their repertoire of detecting molecular although the exact mechanism is not well understood. Our patterns present in pathogens. However, the exact mechanism of studies indicate that differential dimerization of TLR2 can lead differential dimerization of TLR2 by these two PPE proteins re- to fine-tuning of downstream inflammatory cascades involving mains unclear. Interestingly, PPE17 binds to LRR 16–20 domain the PKCε–IRAK3 signaling mediators. (11) and PPE18 binds to LRR 11–15 domain (10) of TLR2, and, by We observed differential redistribution of IRAK3, an inactive virtue of binding to different regions of TLR2 ectodomain, they member of the IRAK family (25), to the cytosol during interaction might recruit different dimers of TLR2. Interestingly, Meng et al. of PPE17 with TLR1/2 versus PPE18 with TLR2/2, a process that (54) have reported that deletion of any region of TLR2 renders it is susceptible to LMB treatment. The fact that TLR1-associated unable to interact with another TLR in presence of a ligand, signaling is indispensable for export of IRAK3 from the nucleus to by guest on September 27, 2021

FIGURE 9. The PPE17-triggered IRAK3 nuclear export and proinflammatory response is critically dependent on TLR1-associated signaling. (A and B) THP-1 macrophages were transfected with either TLR1-specific siRNA or nontarget siRNA, and, after 36 h of transfection, cells were either lysed and immunoblotted with anti-TLR1 Ab (A) or fixed, permeabilized, and stained with anti-IRAK3 Ab, followed by anti-rabbit Alexa Fluor 488 Ab for confocal microscopy (B). Image quantification of nuclear export of IRAK3 in THP-1 macrophages was carried out using ImageJ software. Scale bars, 5mm. About 30 cells per experimental sample were taken for quantification of nuclear export of IRAK3 in THP-1 macrophages (C). THP-1 macrophages were transfected with either TLR1- specific siRNA or scrambled siRNA, and, after 36 h of transfection, cells were used to isolate RNA for measuring TNA-a and b-actin by semiquantitative RT-PCR from the synthesized cDNA (D). Data shown are representative of three independent experiments. 1786 REGULATION OF TLR2 SIGNALING BY PPE PROTEINS the cytosol and induction of proinflammatory cascades in PPE17- PPE18-treated macrophages. The role of MKP-1 to dictate PPE17- treated macrophages is confirmed by the results that silencing of induced MAPK signaling and proinflammatory responses was con- TLR1 inhibits IRAK3 export and TNF-a cytokine production firmed by using MKP-1–specific siRNA that showed decreased upon PPE17 treatment. Interestingly, human TLR1 deficiency was ERK1/2 activation and TNF-a production, but increased p38MAPK found to be associated with reduced inflammation due to decreased activation. This indicated that MKP-1 has an important role to play NF-kB activity and conferred protection from leprosy reversal re- in regulation of MAPK pathway downstream of TLR2. Although action (55). Once PPE17 interacts with the TLR1/2 heterodimer, in the current study we demonstrate an important role of PKCε– MyD88 and PKCε werefoundtoberecruitedtothereceptor IRAK3–MKP-1 signaling in regulating the TLR2-induced MAPK complex, and this probably allowed interaction of PKCε with activation and cytokine responses, various reports suggest that IRAK1. PKCε then translocates to the nucleus through its NLS and TLR2- and TLR4-mediated intracellular signaling pathways prob- is dependent on the IRAK1 because pharmacological inhibitor of ably use differential IRAK3 response (26, 27, 57, 58). In summary, IRAK1/4 activity significantly abrogated nuclear translocation of we propose that PPE17 binding to TLR2 leads to the formation of PKCε. Thus, PKCε appearstobeatargetofIRAK1,andtheclose TLR1/2 heterodimer, causing increased interaction of MyD88, proximity of the two molecules is probably facilitated by MyD88. PKCε, and IRAK1, facilitating nuclear translocation of PKCε. Interestingly, MyD88-deficient mice were found to be susceptible Once inside the nucleus, PKCε phosphorylates IRAK3, which to M. tuberculosis infection and displayed impaired production probably is important for export of nuclear IRAK3 to the cytoplasm. of Th1-type cytokines, indicating an important role of MyD88 in Cytoplasmic IRAK3 then probably causes the activation of ERK1/2 the activation of mycobacteria-induced proinflammatory signaling and stabilization of MKP-1, leading to the activation of proinflamma- cascades (56). Our data suggest that MyD88 probably regulates tory signaling cascades in PPE17-treated macrophages. To the best of Downloaded from inflammatory responses by regulating IRAK1–PKCε signaling our knowledge, this is the first time we report that a PPE protein of events downstream of TLR2. Importantly, it was observed that the M. tuberculosis interacts with TLR1/2 heterodimer to trigger proin- kinase activity of PKCε was essential for the export of IRAK3 from flammatory response via PKCε–IRAK3 signaling pathway. the nucleus to the cytoplasm. The export of IRAK3 from the nu- TLRs have the capacity to determine the Th1/Th2 balance in face cleus to the cytoplasm was found to be diminished in PPE17-treated of an intracellular infection with M. tuberculosis (10, 13, 20, 59, 60).

macrophages when either the PKCε expression was silenced or the The bacillus tries to tip this balance in its favor by modulating the http://www.jimmunol.org/ activity of IRAK1 was inhibited, indicating that probably the host innate immune signaling using ligands such as PPE proteins. IRAK1–PKCε signaling axis has an important role in the export of We have shown earlier that PPE18 skews the Th responses more IRAK3 from the nucleus to the cytoplasm. biased toward an antimycobacterial Th2 type and is involved in the Cytoplasmic IRAK3 is probably responsible for activation of virulence of M. tuberculosis in mice (10, 61). Thus, the M. tuber- ERK1/2, as we observed higher amount of phospho-ERK1/2 in culosis probably modulates the innate immune responses toward PPE17-treated macrophages as compared with PPE18-treated the anti-inflammatory type by avoiding the PKC–IRAK3 signaling macrophages, and pretreatment of PPE17-treated cells with LMB cascade by triggering TLR2/2 homodimerization, and thus inducing or silencing of PKCε expression by PKCε-specific siRNA, which sustained activation of p38MAPK and IL-10 production. It will be prevents nuclear export of IRAK3, resulted in decreased ERK1/2 interesting to investigate whether other ligands for TLR2, which by guest on September 27, 2021 activation. This indicates that the export of nuclear IRAK3 to the induce anti-inflammatory cascades similar to PPE18, also trigger cytosol is crucial for activation of ERK1/2 in PPE17-treated mac- TLR2 homodimerization and activation of p38MAPK. Further- rophages. However, we are yet to understand the mechanism by more, it would be interesting to investigate the role of IRAK3 in which ERK1/2 is activated upon export of nuclear IRAK3 into the regulating inflammation signaling pathways. Our study thus points cytoplasm. It is possible that IRAK3 activates some other signaling out how the TLR2-signaling cascades can be influenced by various intermediates yet to be identified to favor ERK1/2 activation. It has ligands to modulate the macrophage-innate immune responses, been recently reported that ERK1/2 phosphorylates MKP-1 and which may be helpful in improving protective immune response leads to its stabilization (37), and MKP-1 is known to have a higher against not only M. tuberculosis, but also other intracellular path- affinity to deactivate p38MAPK than ERK1/2 (38). This would ogens that are known to modulate macrophage innate cytokine probably explain the pattern of p38MAPK activation that we ob- balance by targeting the TLR2. served with the two PPE proteins. PPE17 treatment led to higher export of nuclear IRAK3 to the cytoplasm, resulting in increased Acknowledgments activation of ERK1/2 and stabilization of MKP-1, which was re- We thank Dr. Shiny Nair and Dr. K. H. Bhat and Niteen Pathak for tech- sponsible for decreased phospho-p38MAPK level in these cells. nical help in purification of PPE17 and PPE18 proteins. As PPE18 fails to trigger significant IRAK3 export from the nucleus to the cytosol, MKP-1 undergoes rapid degradation by the proteasomal machinery, and an increased p38MAPK activity is Disclosures observed in such situation, resulting in poorer ERK1/2 activity The authors have no financial conflicts of interest. (10). The role of IRAK3 in the stabilization of MKP-1 in PPE17- treated cells was confirmed by the siRNA-mediated silencing of References IRAK3 that resulted in poorer MKP-1 level in these cells. The 1. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. induction of TNF-a by PPE17 was dependent upon ERK1/2 ac- Rev. Immunol. 20: 197–216. tivity as well as the export of nuclear IRAK3 to the cytoplasm as 2. Takeuchi, O., and S. Akira. 2010. Pattern recognition receptors and inflamma- a tion. Cell 140: 805–820. treatment of cells with PD98059 as well as LMB reduced TNF- 3. Medzhitov, R., P. Preston-Hurlburt, and C. A. Janeway, Jr. 1997. A human ho- induction in these cells. That MKP-1 was responsible for inhibi- mologue of the Drosophila Toll protein signals activation of adaptive immunity. tion of activated p38MAPK in PPE17-treated macrophages was Nature 388: 394–397. 4. Akira, S. 2006. TLR signaling. Curr. Top. Microbiol. Immunol. 311: 1–16. confirmed when these macrophages exhibited increased p38MAPK, 5. Rosadini, C. V., and J. C. Kagan. 2015. Microbial strategies for antagonizing but decreased ERK1/2 phosphorylation upon LMB treatment that Toll-like-receptor signal transduction. Curr. Opin. Immunol. 32: 61–70. 6. Patterson, N. J., and D. Werling. 2013. To con protection: TIR-domain con- caused poorer export of nuclear IRAK3 in the cytosol, resulting in taining proteins (Tcp) and innate immune evasion. Vet. Immunol. Immunopathol. decreased MKP-1 level, similar to the phenotype displayed by 155: 147–154. The Journal of Immunology 1787

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