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 Supplementary http://www.jimmunol.org/content/suppl/2016/07/30/jimmunol.150181 Material 6.DCSupplemental References This article cites 61 articles, 22 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/197/5/1776.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 heterodime- rization 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 re- sponses are dictated downstream of the TLR2 are not well un- derstood. 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