The PPE18 of Mycobacterium tuberculosis Inhibits NF-κB/rel−Mediated Proinflammatory Production by Upregulating and Phosphorylating This information is current as Suppressor of Cytokine Signaling 3 Protein of September 28, 2021. Shiny Nair, Akhilesh Datt Pandey and Sangita Mukhopadhyay J Immunol 2011; 186:5413-5424; Prepublished online 30 March 2011; Downloaded from doi: 10.4049/jimmunol.1000773 http://www.jimmunol.org/content/186/9/5413 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/03/30/jimmunol.100077 Material 3.DC1 References This article cites 85 articles, 45 of which you can access for free at: http://www.jimmunol.org/content/186/9/5413.full#ref-list-1

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The PPE18 Protein of Mycobacterium tuberculosis Inhibits NF-kB/rel–Mediated Proinflammatory Cytokine Production by Upregulating and Phosphorylating Suppressor of Cytokine Signaling 3 Protein

Shiny Nair, Akhilesh Datt Pandey, and Sangita Mukhopadhyay

Mycobacterium tuberculosis bacteria are known to suppress proinflammatory like IL-12 and TNF-a for a biased Th2 response that favors a successful infection and its subsequent intracellular survival. However, the signaling pathways targeted by the bacilli to inhibit production of these cytokines are not fully understood. In this study, we demonstrate that the PPE18 protein of M. tuberculosis inhibits LPS-induced IL-12 and TNF-a production by blocking nuclear translocation of p50, p65 NF-kB, and c- rel transcription factors. We found that PPE18 upregulates the expression as well as tyrosine phosphorylation of suppressor of Downloaded from cytokine signaling 3 (SOCS3), and the phosphorylated SOCS3 physically interacts with IkBa–NF-kB/rel complex, inhibiting phosphorylation of IkBa at the serine 32/36 residues by IkB kinase-b, and thereby prevents nuclear translocation of the NF-kB/ rel subunits in LPS-activated macrophages. Specific knockdown of SOCS3 by small interfering RNA enhanced IkBa phosphor- ylation, leading to increased nuclear levels of NF-kB/rel transcription factors vis-a-vis IL-12 p40 and TNF-a production in macrophages cotreated with PPE18 and LPS. The PPE18 protein did not affect the IkB kinase-b activity. Our study describes http://www.jimmunol.org/ a novel mechanism by which phosphorylated SOCS3 inhibits NF-kB activation by masking the phosphorylation site of IkBa. Also, this study highlights the possible mechanisms by which the M. tuberculosis suppresses production of proinflammatory cytokines using PPE18. The Journal of Immunology, 2011, 186: 5413–5424.

he macrophages elicit various antimycobacterial mecha- inversely correlated with the levels of IL-12 and TNF-a secreted nisms during the innate phase of activation that play (11–14). Human monocyte-derived macrophages (MDMs) are crucial roles in deciding the outcome of Mycobacterium shown to produce a significantly lower amount of IL-12 after

T by guest on September 28, 2021 tuberculosis infection (1). Among these, production of proinflam- exposure to M. tuberculosis than other bacteria (9), indicating that matory cytokines like IL-12 and TNF-a is critical for mounting an M. tuberculosis specifically targets the signaling cascades that lead optimal defense against mycobacterial infection (2–5). The TNF- to production of this cytokine. However, the molecular mecha- a is important for granuloma formation and induction of cyto- nisms by which the pathogenic mycobacteria can downregulate toxicity against the bacilli (6, 7). In contrast, IL-12 plays a crucial induction of these proinflammatory cytokines are not fully role in activating the protective Th1 immune response (5, 8, 9). understood. Regulation of proinflammatory responses often involves diverse The mycobacteria are characterized by the presence of two signaling cascades, and the pathogenic M. tuberculosis bacteria unique families, the proline-glutamic acid and the proline- have evolved several mechanisms to modulate these signaling proline-glutamic acid (PPE) (15), which are highly expanded in pathways to suppress IL-12 and TNF-a production to favor its pathogenic species like M. tuberculosis (16). Although the long-term survival and persistence inside the host (10–12). In- are predicted to play a role in generating antigenic variation terestingly, virulence of pathogenic mycobacteria is found to be (17–20), the functional role of these proteins in mycobacterial path- ogenesis is not clearly understood. Some of these are pre- dicted to play an important role in mycobacterial persistence inside Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnos- the host and are crucial in M. tuberculosis pathogenesis (21–23). tics, Nampally, Hyderabad 500001, Andhra Pradesh, India Recently, we reported that a PPE family of proteins of M. tu- Received for publication March 8, 2010. Accepted for publication January 28, 2011. berculosis, PPE18, inhibits the Th1-type response by specifically This work was supported by a research fellowship from the Council of Scientific and downregulating IL-12 p40 and other proinflammatory indicators Industrial Research, India (to S.N.), grants from the Department of Biotechnology, Government of India, and a core grant to the Centre for DNA Fingerprinting and (24). We found that PPE18 binds to the TLR2 leucine-rich repeat Diagnostics by the Department of Biotechnology. (LRR) 11–15 region and causes an inhibition of IL-12 p40 (24). It Address correspondence and reprint requests to Dr. Sangita Mukhopadhyay, Labo- appears that PPE18 probably targets the proinflammatory signal- ratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics, ing downstream of TLR2 by activating certain negative regulators Building 7, Gruhakalpa, 5-4-399/B, Nampally, Hyderabad 500001, Andhra Pradesh, India. E-mail address: [email protected] of this pathway. Interestingly, suppressor of cytokine signaling 3 The online version of this article contains supplemental material. (SOCS3) downstream of TLR2 (25) acts as negative regulator of Abbreviations used in this article: EIA, enzyme immunoassay; IKK, IkB kinase; proinflammatory signaling (26–29). Because several pathogenic LRR, leucine-rich repeat; MDM, monocyte-derived macrophage; PPE, proline- mycobacterial species induce expression of the SOCS3 protein proline-glutamic acid; PTK, protein tyrosine kinase; siRNA, small interfering (30–32), we speculated that probably SOCS3 protein is involved RNA; SOCS3, suppressor of cytokine signaling 3. in the suppression of induction of IL-12 p40 in macrophages Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 treated with PPE18. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000773 5414 SOCS3 REGULATES NF-kB/rel SIGNALING IN TUBERCULOSIS

The SOCS3 protein is known to play important roles in the Cytokine assay regulation of proinflammatory responses during infection (25–28, The IL-12 p40 and TNF-a cytokines (BD Pharmingen, San Diego, CA) in 33, 34). Constitutively expressed SOCS3 was found to favor de- the macrophage culture supernatants were quantified by two-site sandwich velopment of Th2-type response and correlate well with the se- EIA as described earlier (43). Standard curves for these cytokines were verity of Th2-mediated disease like atopic asthma (35). Further, obtained using recombinant standard proteins provided along with the kits. SOCS3-transduced dendritic cells are found to be effective in- Total RNA isolation and RT-PCR for SOCS3 ducers of Th2-type T cells in vitro and in vivo through reduced production of IL-12, IFN-g, and IL-23 p19 cytokines (36). Al- RT-PCR was carried out as described elsewhere (24) using a forward primer (59-ATGGTCACCCACAGCAAGTTT-39) and a reverse primer (59- TCA- though the mechanisms are not clear, SOCS3-transduced dendritic CTGGATGCGCAGGTTC-39) specific to SOCS3 (44). The amplification cells are shown to be effective inducers of Th2-type T cells in conditions for SOCS3 and b-actin were as follows: denaturation at 94˚C vitro and in vivo (36). Various studies have indicated a direct role for 30 s, annealing at 58˚C for 1 min, and extension at 72˚C for 2 min. of SOCS3 in downregulating the IFN-g signaling pathway (37, After 30 cycles, the amplified products SOCS3 (293 bp) and b-actin (510 38). SOCS3 is known to predominantly target the STAT family of bp) were resolved by electrophoresis on 1.2% agarose gels and visualized by ethidium bromide staining. proteins to attenuate IFN-g–specific proinflammatory signaling (31, 39, 40). But it could be possible that the effect of SOCS3 is Flow cytometric evaluation of phosphorylated IkBa and Src not restricted to STAT signaling alone but also to other signaling kinase pathways to downregulate IL-12 and TNF-a cytokines during The amount of phosphorylated IkBa in macrophages was measured by tuberculosis. Although mycobacteria are known to increase SOCS3 flow cytometry as described elsewhere (24, 45) using rabbit anti–phospho- expression in macrophages (31, 32), their significance in the IkBa Ab (Cell Signaling Technology, Danvers, MA) followed by in- Downloaded from cubation with anti-rabbit–FITC conjugate (Sigma-Aldrich). The amount of macrophage innate-effector response in tuberculosis is not well 416 understood. It is possible that the bacilli exploit the SOCS3 sig- phosphorylated Src was detected using mouse anti–phospho-Src (Tyr ) (Millipore, Temecula, CA) and detected with anti-mouse-FITC conjugate naling pathway to inhibit proinflammatory cytokine induction in (Sigma-Aldrich). Flow cytometric analysis of the FITC-stained cells was macrophages. carried out on a BD FACSVantage SE flow cytometer (BD Biosciences). In this study, we describe a unique signaling pathway respons- Transient transfection ible for the SOCS3-mediated downregulation of proinflammatory http://www.jimmunol.org/ cytokines that involves the IkBa–NF-kB/rel signaling and conclude PMA-differentiated macrophages were transfected with a Socs3-over- that PPE18 targets this signaling pathway to suppress induction of expressing construct (pCDNA-myc/SOCS3, a kind gift from Prof. K.N. IL-12 p40 and TNF-a cytokines in activated macrophages to favor Balaji, Indian Institute of Science, Bangalore, India) using Lipofectamine 2000 (Invitrogen). The expression vector without any insert (pCDNA/myc) a Th2-type response (24) that helps its replication and persistence of was used as a negative control. After 36 h of transfection, cells were the bacilli inside the hosts. harvested and lysed to prepare total protein extracts. The expression of SOCS3 protein was determined by Western blotting using anti-SOCS3 Ab. Materials and Methods Preparation of nuclear extracts and Western blot analysis Expression and purification of rPPE18 protein Nuclear extracts and cytoplasmic extracts were prepared as described ear- by guest on September 28, 2021 The rPPE18 protein was expressed and purified as described earlier (24). lier (46, 47). Equal amounts of cytoplasmic or nuclear extracts from var- The purified recombinant proteins were dialyzed against several changes ious groups were separated by 12% SDS-PAGE. The p50, p65, and c-rel of PBS. The protein concentration was determined using a kit that employs levels were measured in both cytoplasmic and nuclear extracts, whereas the bicinchoninic acid method of protein estimation (Pierce, Rockford, IL) total IkBa level was determined in cytoplasmic extracts. Following elec- and was incubated with 10% v/v polymyxin B-agarose (Sigma-Aldrich, St. trophoretic transfer, the nitrocellulose membranes were incubated with Louis, MO) (41). The purified protein had a very low endotoxin content rabbit Ab to p50, p65, c-rel, or IkBa protein (Santa Cruz Biotechnology, (,0.05 EU/ml) as measured by the E-toxate (Limulus amebocyte lysate) Santa Cruz, CA). The membranes were next incubated with anti-rabbit kit (Sigma-Aldrich) and was used for all in vitro experiments. Ig-HRP conjugate (Sigma-Aldrich). Bound reporter enzyme was detected by ECL kit following the manufacturer’s protocol (ECL; GE Healthcare, Cell culture Little Chalfont, U.K.). In some experiments, equal loading of protein was b The human monocytic cell line THP-1 was obtained from the National confirmed by measuring the -actin level in the same extracts by Western Centre for Cell Science, Pune, India. The suspension cell line was main- blotting. tained in RPMI 1640 medium (Invitrogen) supplemented with 10% (v/v) EMSA for NF-kB heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (all from Invitrogen) at 37˚C in a 5% CO2 humidified Nuclear extracts (10 mg) prepared from various experimental groups were incubator. The THP-1 cells were differentiated to macrophages by in- incubated for 30 min at room temperature with 1 ng [32P] end-labeled du- cubation with 20 ng/ml PMA (Sigma-Aldrich) for 24 h followed by a rest plex oligodeoxyribonucleotide containing the NF-kB binding region, (59-TTG- for overnight in media containing 1% FBS. Human PBMCs obtained from TTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-39 Lonza (Walkersville, MD) were diluted in RPMI 1640 medium (Invi- and 59-CCACGCCTCCCTGGAAAGTCCCCAGCGGAAAGTCCCTTG- trogen) containing 10% FBS and seeded in 100-mm petri dish at a density TAACAA-39), and EMSA was performed as described earlier (41). of 20 3 106 cells. The MDMs were purified as described elsewhere (42). Reporter assay Macrophage activation assay To determine the NF-kB activity in various experimental groups, we used PMA-differentiated THP-1 macrophages or human MDMs were treated in a luciferase NF-kB reporter construct. The pSV–b-galactosidase plasmid triplicate with either increasing concentrations of rPPE18 or a fixed con- (Promega) was used to normalize transfection efficiency. The PMA- centration (3 mg/ml) of rPPE18 protein in the absence or presence of 3 mg/ differentiated THP-1 macrophages were cotransfected with either 3xIgk- ml bacterial LPS (Sigma-Aldrich) (24). In some experiments, tyrosine ConA-Luc (NF-kB–luciferase reporter plasmid) and pSV–b-galactosidase kinase inhibitor genistein (100 mM) (Merck-Calbiochem, Darmstadt, Ger- plasmid or pConA (vector control) and pSV–b-galactosidase plasmid us- many), p38 MAPK-specific inhibitor, SB203580, or known Src kinase ing Lipofectamine 2000 reagent (Invitrogen). After 24 h of transfection, inhibitors like SU6656 or PP2 (AG 1879) or Src Inhibitor-1 (all purchased cells were either left untreated or pretreated with rPPE18 (3 mg/ml) for 1 h from Sigma Aldrich) were used. Cells were pretreated with SB203580 or followed by stimulation with LPS (3 mg/ml) for another 1 h. Cells were Src kinase inhibitors 1 h before incubation with rPPE18. Cells were har- lysed and assayed for luciferase activity using luciferase reporter assay vested at various time points or after 1 h to isolate total RNA or for pre- system according to the instructions of the manufacturer (Promega), and paration of protein extracts or cultured for 48 h for estimating IL-12 p40 luciferase activity was monitored with a luminometer (Turner Designs, and TNF-a levels in the culture supernatants by enzyme immunoassay Sunnyvale, CA). The b-galactosidase expression was measured by b-ga- (EIA). lactosidase ELISA as per the manufacturer’s protocol (Roche Diagnostics). The Journal of Immunology 5415

Luciferase activity values were normalized to transfection efficiency mea- were transiently cotransfected with the NF-kB luciferase plasmid sured by b-galactosidase assay (Roche Diagnostics). The results are along with a b-galactosidase reporter construct driven by a con- expressed as relative NF-kB activity of the test samples compared with stitutive promoter to normalize the luciferase activity. At 24 h that of the pConA-transfected samples after normalizing for b-galactosi- dase activity and protein concentration. posttransfection, these macrophages were stimulated with 3 mg/ml LPS in the absence or presence of 3 mg/ml rPPE18 protein. To our Silencing SOCS3 expression using SOCS3-specific small expectation, the rPPE18 protein was found to attenuate LPS- interfering RNA stimulated NF-kB activity (Fig. 1A)by∼2-fold. To further ver- For generating small interfering RNA (siRNA), primers were designed as ify these observations, we measured the cognate DNA-binding follows: 59-ATCCGCCAAGAACCTGCGCATCCATTCAAGGAGATGGA- activity of NF-kB by EMSA. The PMA-differentiated THP-1 TGCGCAGGTTCTTGGTTTTTG-39 and 59-AATTCAAAAAACCAAGAA- macrophages were preincubated with either medium alone or CCTGCGCATCCATCTCCTTGAATGGATGCGCAGGTTCTTGGCG-39, rPPE18 protein and then stimulated with LPS. Nuclear extracts and scramble oligonucleotides were used from custom SMARTPools from Dharmacon (Lafayette, CO). These oligonucleotides were annealed and were prepared, and the DNA-binding activity of NF-kB was de- ligated into pSIREN plasmid in BamH1 and EcoR1 sites and confirmed by termined by EMSA using duplex oligonucleotides containing NF- sequencing. These constructs were used to transfect PMA-differentiated kB binding sites. DNA-binding activity of NF-kB was found to be THP-1 macrophages using Lipofectamine 2000 reagent (Invitrogen). low in unstimulated macrophages or treated with rPPE18 alone IkB kinase activity assay (Fig. 1B, lanes 2 and 4). The DNA-binding activity of NF-kB was found to be highest in macrophages stimulated with LPS; how- IkB kinase (IKK) activity was measured using the IKK-b inhibitor ever, the complex formation was markedly reduced in LPS- screening kit (Merck-Calbiochem). Approximately 100 ng GST-IkBa stimulated cells treated with rPPE18 (Fig. 1B, compare lane 5 Downloaded from substrate was incubated with either 5 ng human rIKK-b (provided by the manufacturer) or IKK-a/b was pulled down from macrophage lysates (500 with lane 3). The specificity of the NF-kB complex was confirmed mg) using rabbit anti–IKK-a/b Ab (2 mg) (Santa Cruz Biotechnology) by the competition assay using unlabeled NF-kB (Fig. 1B, lane 6). bound to the protein G-Sepharose beads (Sigma-Aldrich). In some wells, specific IKK-b inhibitor (provided by the manufacturer) was added. The PPE18 prevents translocation of NF-kB/rel subunits to nucleus reaction mixtures (10 ml) were added to wells precoated with glutathione in LPS-activated macrophages to allow capture of GST-IkBa. The phosphorylated GST-IkBa substrate was detected using the mouse anti–phospho-IkBa (serine 32/36) Ab, fol- The p50 and p65 NF- kB and c-rel are important transcription http://www.jimmunol.org/ lowed by incubation with the anti-mouse IgG-HRP conjugate (Sigma- factors that are known to regulate IL-12 p40 gene transcription Aldrich). The HRP activity was measured using TMB substrate follow- (50, 51, 53) as well as induction of TNF-a (52, 54). Because ing the manufacturer’s protocol. The absorbance was measured at 450 nm PPE18 was found to inhibit NF-kB activity in LPS-stimulated and is directly related to the level of IKK activity. macrophages (Fig. 1), we next examined the nuclear levels of Immunoprecipitation assay p50, p65, and c-rel transcription factors in these macrophages by Western blotting using specific Abs against these transcription PMA-differentiated THP-1 macrophages were treated with 3 mg/ml rPPE18 in the absence or presence of LPS (3 mg/ml). Whole-cell extracts were factors. The Western blot results indicate that nuclear levels of all prepared by using lysis buffer as described earlier (42). The coimmuno- of these NF-kB/rel transcription factors are reduced in macro- precipitated proteins were detected by Western blotting using appropriate phages cotreated with rPPE18 and LPS as compared with the by guest on September 28, 2021 combinations of primary and secondary Abs. macrophages treated with LPS alone (Fig. 2A, bottom panel, Statistical analysis compare lane 4 with lane 2). In contrast, the levels of p50, p65, and c-rel factors were higher in cytoplasm in the groups cotreated Data were expressed as mean 6 SD of three independent experiments performed with similar results. The data were analyzed using Student t test with rPPE18 and LPS as compared with the group treated with wherever applicable. A p value , 0.05 was considered to be significant. LPS alone (Fig. 2A, top panel, compare lane 4 with lane 2), in- dicating that PPE18 protein probably inhibits the translocation of NF-kB/rel transcription factors from cytoplasm to nucleus. Den- Results sitometric analyses were also carried out using AlphaEaseFC k The rPPE18 protein attenuates NF- B activity in LPS-treated software and the Spot Denso tool (Version 7.0.1; Alpha Innotech, macrophages San Leandro, CA), and the mean intensity of the bands was pre- The purified rPPE18 protein of M. tuberculosis was found to in- sented as arbitrary unit in a graph (Fig. 2B). hibit LPS-induced proinflammatory cytokines like TNF-a and IL- k a 12 p40 in a dose-dependent manner (Supplemental Fig. 1A). The PPE18 inhibits LPS-induced phosphorylation of I B concentrations at which rPPE18 inhibited TNF-a and IL-12 p40 The NF-kB transcription factors are shown to be controlled up- IC50 were found to be 292 ng/ml 6 64.2 (7.49 pM) and 257.8 ng/ stream by the cytoplasmic inhibitory protein known as IkBa (55– ml 6 21.4 (6.59 pM), respectively. Although the degree of in- 57). IkBa interacts and sequesters p50 and p65 NF-kB and c-rel hibition was maximal in the range of 1–10 mg/ml, for all of our proteins in the cytoplasm and prevents nuclear translocation of subsequent experiments, we used a concentration of 3 mg/ml to p50 and p65 NF-kB and c-rel proteins from cytoplasm (57). Dur- take care of batch-to-batch qualitative variations of the purified ing macrophage activation, phosphorylation of IkBa at serine 32 recombinant protein. We also observed that PPE18 at 3 mg/ml and 36 residues by the IKKs marks it for degradation via ubiq- concentrations could inhibit early induction of TNF-a and IL-12 uitination and consequently releases the NF-kB/rel transcription p40 in LPS-activated macrophages (Supplemental Fig. 1B). factors to translocate to the nucleus, where they activate tran- The NF-kB transcription factors are known to play critical roles scription of the target genes. Because PPE18 is able to inhibit in the induction of proinflammatory cytokines (48, 49), as the LPS-induced NF-kB activity by inhibiting nuclear translocation of promoters of IL-12 p40 (50, 51) and TNF-a (52) contain NF-kB p50 and p65 NF-kB and c-rel transcription factors (Figs. 1, 2), we binding sites, and transcription of these genes is essentially de- next determined whether PPE18 inhibits IkBa phosphorylation, pendent on binding of NF-kB/rel transcription factors to these which is a crucial event for NF-kB translocation and subsequent sites. Therefore, we measured if NF-kB activity was lower in LPS- activation of target genes. It was found that LPS-induced IkBa treated macrophages in the presence of rPPE18 using a luciferase phosphorylation was significantly inhibited in the presence of reporter construct. The PMA-differentiated THP-1 macrophages rPPE18 (Fig. 3A). This was well correlated with the observation 5416 SOCS3 REGULATES NF-kB/rel SIGNALING IN TUBERCULOSIS Downloaded from

FIGURE 1. LPS-induced NF-kB activity is inhibited by rPPE18 in THP-1 macrophages. A, PMA-differentiated THP-1 macrophages were transfected with either the vector control alone (pConA) or the NF-kB luciferase reporter plasmid (3xIgk-ConA-Luc) along with pSV–b-galactosidase plasmid. After 24 h, the macrophages were treated for 1 h with either medium or rPPE18 (3 mg/ml) and then stimulated with LPS (3 mg/ml) for another 1 h. Tran- scriptional activity of NF-kB was measured by luciferase and b-galactosidase assays. Data are shown as fold inductions over basal activity and represented http://www.jimmunol.org/ as mean 6 SD of three independent experiments performed in triplicate. B, Nuclear extracts prepared from PMA-differentiated THP-1 macrophages activated with LPS (3 mg/ml) in the absence or presence of rPPE18 (3 mg/ml) were subjected to EMSA. The data shown are representative of three in- dependent experiments with similar results. that IkBa was poorly degraded in the rPPE18 and LPS cotreated status of the GST-IkBa substrate is reduced (Fig. 3C, compare group compared with the group treated with LPS alone (Fig. 3B, lane 7 with lane 6) to almost the negative control levels (Fig. 3C, compare lane 4 with lane 2). These results indicate that PPE18 lane 1). These data indicate that IKK-b is predominantly involved affects LPS-induced phosphorylation and degradation of IkBa. in the signal-induced phosphorylation of IkBa in these cells. We

Because the IKKs, notably IKK-a and IKK-b, are involved in observed that PPE18 did not affect the IKK-b activity (Fig. 3C) by guest on September 28, 2021 signal-induced phosphorylation of IkBa (58, 59), experiments because there was no difference in the ability of IKK-b to phos- were carried out to explore if PPE18 inhibits IkBa phosphoryla- phorylate GST-IkBa in the absence (Fig. 3C, lane 4) or presence tion by affecting the IKK-a/b activity. Therefore, we pulled down (Fig. 3C, lane 6) of rPPE18. IKK-a and IKK-b from the cell extracts treated with rPPE18 using a rabbit polyclonal anti–IKK-a/b Ab and then measured its PPE18 increases SOCS3 expression in macrophages ability to phosphorylate GST-IkBa substrate in vitro. We found Because mycobacteria are known to increase the levels of SOCS3 that in the presence of the IKK-b inhibitor, the phosphorylation proteins (31, 32), and several studies have implicated a role of

FIGURE 2. PPE18 protein abrogates cytonuclear translocation of NF-kB/rel subunits in macrophages treated with LPS. A, Cytoplasmic (CE) and nuclear (NE) extracts were prepared from PMA-differenti- ated THP-1 macrophages treated with LPS (3 mg/ ml) without or with rPPE18 (3 mg/ml), and levels of p50, p65, and c-rel transcription factors were measured by Western blotting using anti-p50, anti- p65, or anti–c-rel Ab, respectively. Data are repre- sentative of three independent experiments with similar results. B, Graph representing densitometric analysis of blots represented in A. Mean intensity is expressed as arbitrary units of three independent experiments. The Journal of Immunology 5417 Downloaded from http://www.jimmunol.org/

FIGURE 3. rPPE18 protein inhibits LPS-induced phosphorylation of IkBa without affecting IKK-b activity. A, PMA-differentiated THP-1 macrophages were stimulated with LPS (3 mg/ml) in the absence or presence of rPPE18 (3 mg/ml). The phosphorylation of IkBa in various groups at the 15-min time point was analyzed by flow cytometry. B, Total IkBa levels in the cytoplasmic extracts were detected by Western blotting using anti-IkBa Ab. C, Whole- cell extracts were prepared from PMA-differentiated THP-1 macrophages treated with either medium or rPPE18 (3 mg/ml). Equal amounts of total proteins by guest on September 28, 2021 (500 mg) were used to immunoprecipitate IKK-a/b using anti–IKK-a/b bound to protein G-Sepharose beads, and the immunoprecipitated IKK-a/b was used to assess IkBa phosphorylation (mean 6 SD) in the absence or presence of IKK-b inhibitor (provided by the manufacturer) as described in the Materials and Methods. In the positive control, GST-IkBa was incubated with the rIKK-b in the presence of medium alone (lane 2), and the specificity of the assay in lane 2 was confirmed by adding specific IKK-b inhibitor (lane 3). In negative control group, no IKK-b was added (lane 1). Data are rep- resentative of three independent experiments with similar results.

SOCS3 in attenuation of proinflammatory signaling (25–28), IkBa phosphorylation and translocation of NF-kB subunits into we speculated a role of SOCS3 in the observed PPE18-mediated the nucleus and thereby suppresses induction of proinflammatory downregulation of proinflammatory response. Interestingly, an in- cytokines. To validate this hypothesis, we suppressed SOCS3 ex- crease in the SOCS3 mRNA level was observed as early as 1 h pression by using SOCS3-specific siRNA and examined the effect after treatment with rPPE18 (3 mg/ml), and this expression was of PPE18 on IkBa phosphorylation and NF-kB activity in LPS- sustained until 24 h posttreatment (Fig. 4A). The SOCS3 mRNA treated THP-1 macrophages. The nuclear levels of p65 NF-kB and levels were increased by rPPE18 in a dose-dependent manner (Fig. c-rel and induction of IL-12 p40 and TNF-a were also determined. 4B). The mRNA levels of SOCS3 were also found to be well The PMA-differentiated THP-1 macrophages were transfected correlated with the levels of SOCS3 protein (Fig. 4C). We also with either the scrambled siRNA (THP-1 control group) or examined whether PPE18 could increase SOCS3 expression SOCS3-specific siRNA (THP-1–SOCS3i group), and after 24 h when presented in the context of whole bacterium using an M. posttransfection, these macrophages were stimulated with 3 mg/ml smegmatis (that naturally lacks the PPE18 gene) overexpressing LPS in the absence or presence of 3 mg/ml rPPE18. Depletion of PPE18 (M. smegmatis–pMV-PPE18) (24). Interestingly, there was SOCS3 mRNA levels by siRNA was verified by semiquantitative a significant increase in the SOCS3 expression in macrophages RT-PCR as well as by Western blotting at the protein level infected with M. smegmatis–pMV-PPE18 as compared with the (Supplemental Fig. 2A,2B). Silencing of endogenous SOCS3 was macrophages infected with M. smegmatis harboring the pMV261 found to rescue PPE18-mediated inhibition of IkBa phosphory- vector alone (M. smegmatis-pMV) (Fig. 4D). lation in LPS-stimulated THP-1 macrophages (Fig. 5A). Con- comitantly, an increase in the NF- kB activity was also observed in PPE18 targets SOCS3 signaling to downregulate LPS-induced the THP-1–SOCS3i group as compared with the THP-1 control k a k I B phosphorylation and translocation of NF- B/rel subunits group (Supplemental Fig. 3). As expected, a simultaneous up- to nucleus vis-a-vis induction of proinflammatory cytokines regulation in the nuclear p65 and c-rel levels in response to LPS Because we found that PPE18 could increase SOCS3 levels and activation was also observed in THP-1–SOCS3i macrophages in inhibit LPS-induced IkBa phosphorylation, we predicted that the presence of rPPE18 as compared with that of THP-1 control PPE18 probably targets the SOCS3 protein to inhibit LPS-induced macrophages (Fig. 5B). Depletion of SOCS3 also reversed the 5418 SOCS3 REGULATES NF-kB/rel SIGNALING IN TUBERCULOSIS

possible mechanisms can be envisioned. First, the SOCS3 may inhibit the IKK-b activity or alternatively may physically interact with the IkBa–NF-kB/rel complex in the cytoplasm. Because we found earlier that SOCS3 does not affect the IKK-b activity (Fig. 3C), we speculated that SOCS3 probably interacts with the IkBa– NF-kB complex physically and thereby prevents IKK-b to access the phosphorylation sites on IkBa. To validate this premise, we first coimmunoprecipitated SOCS3-interacting protein(s) using anti-SOCS3 Ab from the extracts prepared from the rPPE18- treated THP-1 macrophages and then subjected to Western blot- ting using anti-IkBa Ab as well as Abs to p50, p65, and c-rel transcription factors. Intriguingly, we found that IkBa along with p50, p65, and c-rel transcription factors could be coimmunopre- cipitated using anti-SOCS3 Ab (Fig. 6Ai). Similar results were also obtained in human MDMs (Fig. 6Aii ), indicating that SOCS3 probably interacts physically with the IkBa–NF-kB/rel complex. Reciprocal coimmunoprecipitation experiments were carried out by coimmunoprecipitating cell extracts prepared from rPPE18-

treated THP-1 macrophages using Ab to p50, p65, c-rel, or IkBa Downloaded from followed by Western blotting using anti-SOCS3 Ab. In all of the cases, we found that SOCS3 could be coimmunoprecipitated along with p50, p65, c-rel, and IkBa, respectively (Fig. 6B, upper panel). As input controls, the levels of p50, p65, c-rel, and IkBa in the cell FIGURE 4. rPPE18 protein increases SOCS3 levels in THP-1 macro- extracts from medium and PPE18-treated macrophages were de- phages. PMA-differentiated THP-1 macrophages were either treated with a fixed concentration (3 mg/ml) of rPPE18 for various time points (A)or termined by immunoblotting with appropriate combinations of http://www.jimmunol.org/ with different concentrations of rPPE18 protein for a fixed time of 2 h (B). primary and secondary Ab (Fig. 6B, lower panel). These data in- LPS (3 mg/ml for 2 h) was added as a positive control. RNAs from all these dicate that SOCS3 physically interacts with the IkBa–NF-kB/rel groups were extracted and subjected to semiquantitative RT-PCR for complex that prevents IkBa phosphorylation and its subsequent SOCS3. SOCS3 levels induced by different concentrations of rPPE18 were degradation. These data suggest a novel mechanism by which also checked by Western blot analysis using anti-SOCS3 Ab (C). SOCS3 SOCS3 can participate in the regulation of NF-kB/rel–mediated expression at different time points was checked in PMA-differentiated signaling cascades. THP-1 macrophages infected with either M. smegmatis strain overex- pressing PPE18 (M. smegmatis-pMV-PPE18) or M. smegmatis harboring Tyrosine phosphorylation of SOCS3 is essential for its

the vector alone (M. smegmatis-pMV) at multiplicity of infection of 10 interaction with IkBa–NF-kB/rel complex by guest on September 28, 2021 (D). Data are representative of three to four independent experiments with similar results. Because tyrosine phosphorylation of SOCS3 has been shown to be important for its various interactions with target proteins (61– 64), we evaluated if the observed interaction of SOCS3 with IkBa PPE18-mediated inhibition of IL-12 p40 and TNF-a induction in is due to the phosphorylation of either SOCS3 or IkBa by PPE18. these macrophages (Fig. 5C, compare bar 8 with bar 4; p , 0.001 Therefore, extracts prepared from rPPE18-treated macrophages in both the cases). Further, these results also hint to an additional were immunoprecipitated with anti-SOCS3 Ab and probed with signaling pathway regulating the induction of proinflammatory anti-phosphotyrosine Ab. Interestingly, only a single band corre- cytokines (IL-12 p40 and TNF-a) via the SOCS3 signaling path- sponding to the expected molecular mass of SOCS3 (30 kDa) way. To further substantiate our observation in PMA-differentiated was observed (Fig. 7Ai); however, no band corresponding to the THP-1 macrophages that PPE18 targets the SOCS3 to inhibit molecular mass of IkBa (41 kDa) was detectable (Fig. 7Ai). In nuclear translocation of NF-kB/rel subunits and consequently contrast, when the same blot was stripped and reprobed with anti- inhibits proinflammatory cytokines in LPS-treated macrophages, IkBa Ab, a band corresponding to the expected molecular mass of these experiments were also carried out in human MDMs. De- IkBa was observed (Fig. 7Aii), indicating that IkBa that interacts pletion of endogenous SOCS3 by siRNA in MDMs resulted in with SOCS3 is not tyrosine phosphorylated by PPE18, and PPE18 upregulation of the levels of LPS-induced nuclear c-rel and p65 as predominantly phosphorylates the tyrosine residue(s) of SOCS3. compared with the group that received the scrambled siRNA In another experiment, the same extract was immunoprecipitated (Fig. 5D). This was correlated with concomitant increase of IL-12 with anti-IkBa Ab and probed with either anti-phosphotyrosine p40 and TNF-a induction in these cells (Fig. 5E, compare bar 8 Ab (Fig. 7Aiii) or anti-SOCS3 Ab (Fig. 7Aiv). In line with our with bar 4; p , 0.001 in both the cases). It will be pertinent to earlier observation, tyrosine phosphorylation was only detected in mention in this study that the SOCS3-deficient mice are embry- SOCS3 but not in IkBa (Fig. 7Aiii, iv). Comparable results were onically lethal (60); therefore, the role of SOCS3 in the regulation also obtained in human MDMs (Fig. 7Bi–iv). These results sug- of macrophage response is most widely studied using SOCS3- gest that rPPE18 not only increases the expression of SOCS3 but specific siRNA construct to knockdown its expression in vitro also triggers tyrosine phosphorylation of SOCS3, which in turn (25, 33). interacts with IkBa. When the phosphorylation status of the immunoprecipitated SOCS3 was assessed in THP-1 macrophages k a k SOCS3 physically interacts with I B –NF- /B/rel complex treated with various concentrations of rPPE18, a dose-dependent Because PPE18 targets the SOCS3 protein to suppress LPS- increase in tyrosine phosphorylation in addition to upregulation of induced proinflammatory responses, our next aim was to decode SOCS3 protein expression was observed (Supplemental Fig. 4). the possible mechanism(s) by which PPE18 elicits such an anti- To further address whether tyrosine phosphorylation of SOCS3 is inflammatory response involving SOCS3. In this regard, two essential for its interaction with IkBa–NF-kB/rel complex, we The Journal of Immunology 5419 Downloaded from http://www.jimmunol.org/

FIGURE 5. PPE18 protein targets the SOCS3 protein to inhibit NF-kB/rel activity and proinflammatory cytokines in LPS-treated macrophages. A, The by guest on September 28, 2021 PMA-differentiated THP-1 macrophages were transiently transfected with scrambled siRNA (THP-1-control group) or SOCS3-specific siRNA (THP-1– SOCS3i group), and after 24 h, cells were stimulated with 3 mg/ml LPS in the presence of medium or rPPE18 (3 mg/ml). Cells were harvested either after 15 min to measure IkBa phosphorylation by flow cytometry or after 1 h to measure nuclear levels of c-rel and p65 transcription factors by Western blotting (B) or cultured for 48 h to measure IL-12 p40 and TNF-a by EIA (C). D, Human MDMs transfected with either scrambled siRNA (MDM-control group) or SOCS3-specific siRNA (MDM-SOCS3i group) stimulated with 3 mg/ml LPS in the presence of medium or rPPE18 (3 mg/ml). Cells were either harvested after 1 h to measure nuclear levels of c-rel and p65 by Western blotting or cultured for 48 h to measure IL-12 p40 and TNF-a by EIA (E). Data are representative of three independent experiments with similar results preincubated THP-1 macrophages with genistein (a known in- very high levels of SOCS3 were present in the THP-1 macro- hibitor of tyrosine phosphorylation) and then incubated with 3 mg/ phages transfected with pCDNA-myc/SOCS3 (Fig. 7D, upper ml rPPE18 and examined SOCS3–IkBa interaction by coimmu- panel, lane 2), in the absence of rPPE18, no IkBa bound to noprecipitation. We observed that genistein prevented tyrosine SOCS3 could be immunoprecipitated with anti-SOCS3 Ab (Fig. phosphorylation of SOCS3 in rPPE18-treated macrophages (Fig. 7D, lower panel, lane 2). However, in the presence of rPPE18, 7Ci, compare lane 4 with lane 2), and no IkBa could be immu- IkBa was immunoprecipitated using the same amount of anti- noprecipitated using anti-SOCS3 Ab when these macrophages SOCS3 Ab (Fig. 7D, lower panel, lane 3). These results clearly were treated with rPPE18 in the presence of genistein (Fig. 7Cii, indicate that IkBa does not interact with the SOCS3 that is not compare lane 4 with lane 2). Genistein treatment had no direct tyrosine phosphorylated. Because our earlier studies revealed that effect on the total IkBa levels (Fig. 7Ciii) or on the SOCS3 in- the N-terminal domain of PPE18 is primarily responsible for its duction by rPPE18 (Fig. 7Civ). These results indicate that tyrosine interaction with TLR2 (24), we next investigated the domain of phosphorylation of SOCS3 is essential for its physical interac- PPE18 involved in mediating tyrosine phosphorylation SOCS3. tion with IkBa. To further substantiate this observation, SOCS3 The THP-1 macrophages were treated with the full-length or N- protein was transiently overexpressed by transfecting THP-1 mac- terminal truncated or C-terminal truncated PPE18 protein and rophages with SOCS3 overexpression plasmid (pCDNA-myc/ checked for tyrosine phosphorylation of SOCS3. The coimmu- SOCS3) and examined whether constitutively expressed SOCS3 noprecipitation data indicate that the C-terminal truncated PPE18 could also interact in vivo with IkBa–NF-kB/rel complex. THP-1 could induce SOCS3 tyrosine phosphorylation as efficiently as the macrophages transfected with the backbone vector (pCDNA-myc) full-length PPE18 protein, whereas no SOCS3 tyrosine phos- served as the negative control group. In the positive control group, phorylation could be detected in macrophages treated with N- THP-1 macrophages were treated with rPPE18 (3 mg/ml). The terminal truncated PPE18 protein. These data indicate that the lysates prepared from these groups were used to immunoprecipi- N-terminal domain of PPE18 is important in mediating tyrosine tate the SOCS3–IkBa complex using anti-SOCS3 Ab. Although phosphorylation of SOCS3 (Supplemental Fig. 5). 5420 SOCS3 REGULATES NF-kB/rel SIGNALING IN TUBERCULOSIS

FIGURE 6. SOCS3 physically interacts with IkBa– NF-kB/rel complex. A, The PMA-differentiated THP-1 macrophages (i) or human MDMs were treated with either medium or rPPE18 (ii)(3mg/ml). After 1 h, cells were harvested, and whole-cell lysates were prepared. Cell lysates were immunoprecipitated (IP) with goat anti-SOCS3 Ab, and precipitates were immunoblotted with rabbit anti-p50, anti-p65, anti–c-rel, or anti-IkBa Ab. The total SOCS3 level in the whole-cell lysates was checked by immunoblotting (WB) using goat anti- SOCS3 Ab. B, Reciprocal coimmunoprecipitation ex- periments were carried out wherein whole-cell lysates from the PMA-differentiated THP-1 macrophages treated with either medium alone or rPPE18 (3 mg/ml) were immunoprecipitated with rabbit anti-p50, anti- p65, anti–c-rel, or anti-IkBa Ab and immunoblotted using goat anti-SOCS3Ab. As input controls, the levels Downloaded from of p50, p65, c-rel, and IkBa in the cell extracts from medium, and PPE18-treated macrophages were deter- mined by immunoblotting with appropriate combinations of primary and secondary Ab. Blots are representative of three to four independent experiments with similar results. http://www.jimmunol.org/

PPE18 induces expression of SOCS3 and its phosphorylation Src kinase activation is required for tyrosine phosphorylation of at tyrosine residue(s) by targeting p38 MAPK and Src family SOCS3 in PPE18-treated THP-1 macrophages. We used known kinase(s), respectively selective Src-family PTK inhibitors like PP2, SU6656, and Src

Several reports have implicated a prominent role of p38 MAPK inhibitor-1 (71–73). The THP-1 macrophages were pretreated with by guest on September 28, 2021 signaling in SOCS3 induction (32, 65–67). In our previous study, these inhibitors for 1 h and then treated with 3 mg/ml rPPE18, and we found that PPE18 induces early activation of p38 MAPK, SOCS3 tyrosine phosphorylation was examined. It was observed which is in turn necessary for the IL-10 induction via TLR2 (24). that PP2 (5 and 10 mM) as well as SU6656 (5 and 10 mM) and Src In light of these observations, it was interesting to investigate the inhibitor-1 (1 and 5 mM) could inhibit PPE18-induced SOCS3 importance of p38 MAPK in PPE18-mediated SOCS3 expression. tyrosine phosphorylation (Fig. 8C). These results indicate a pos- Therefore, we pretreated THP-1 macrophages with SB203580 (2.5 sible role of the Src family kinases in the PPE18-mediated tyro- and 5 mg/ml concentration), a specific inhibitor of the p38 MAPK sine phosphorylation of SOCS3. Therefore, it appears that PPE18 pathway (24), before exposing them to rPPE18, and SOCS3 induces SOCS3 expression by activating the p38 MAPK and mRNA expression was measured by semiquantitative RT-PCR. SOCS3 phosphorylation by activating the Src kinases. We found that SB203580 decreased SOCS3 in PPE18-treated macrophages in a concentration-dependent manner Discussion (Fig. 8A). These results suggest the fact that induction of SOCS3 During the innate phase of immune response, the macrophage- by PPE18 probably depends on its activation of the upstream p38 secreted cytokines IL-12 and TNF-a play major roles in the de- MAPK. velopment of Th1 T cells and activation of cell-mediated immune To understand the mechanism through which PPE18 could also responses (2, 3). These proinflammatory cytokines are vital com- induce tyrosine phosphorylation of SOCS3, we evaluated the role ponents of the host defense in response to intracellular infection of protein tyrosine kinases (PTKs), especially the Src family ty- like tuberculosis and therefore are targeted at multiple points by rosine kinases. Increasing evidence suggests that members of the the pathogenic mycobacteria to avoid immune-mediated clearance Src family of PTKs play important roles in tyrosine phosphory- (9, 10). Thus, identification of hijacking points that prevent in- lation of SOCS3 (68, 69). Therefore, we first investigated whether duction of these cytokines during mycobacterial infection is im- Src PTKs are activated in the macrophages that were treated with portant to device an appropriate immune intervention to circumvent rPPE18 by analyzing the Src PTKs phosphorylation status using mycobacterial infection. the anti–phospho-Src family Ab (p-Tyr416). It has been shown that The mycobacterial species are known to upregulate SOCS3 Tyr416 residue of Src can be phosphorylated, and this is important expression (31, 32), and SOCS3 has been shown to specifically for upregulation of Src kinase activity (70). Therefore, we ex- inhibit signaling by IFN-g, the IL-6 family of cytokines, and other amined whether PPE18 can induce Src kinase activity in THP-1 molecules (27, 38, 74). Several groups have reported that myco- macrophages by assessing the phosphorylaton status of Src kinase bacterial infection could increase the levels of SOCS3 protein that by flow cytometry. We found that Src got phosphorylated in inhibit IFN-g–stimulated JAK/STAT signaling and consequently PPE18-treated THP-1 macrophages as early as 15 min and was inhibit proinflammatory gene expression (31). Further, ectopic ex- increased up to 60 min (Fig. 8B). Next, we investigated whether pression of SOCS3 in macrophage at physiological levels was The Journal of Immunology 5421 Downloaded from http://www.jimmunol.org/

FIGURE 7. rPPE18 induces SOCS3 tyrosine phosphorylation, and tyrosine-phosphorylated SOCS3 interacts with IkBa–NF-kB/rel complex. PMA- differentiated THP-1 macrophages (A) or human MDMs (B) were treated with 3 mg/ml of rPPE18 for 1 h. The cell lysates were immunoprecipitated (IP) with either goat anti-SOCS3 Ab (i, ii) or rabbit anti-IkBa Ab (iii, iv), and the precipitates were immunoblotted (WB) with either mouse anti-phospho- tyrosine Ab (i, iii), rabbit anti-IkBa Ab (ii), or with goat anti-SOCS3 Ab (iv). C, In another experiment, PMA-differentiated THP-1 macrophages were treated with 3 mg/ml rPPE18 in the presence of vehicle control (DMSO) or genistein (100 mM). After 1 h, cells were harvested, and whole-cell lysates were prepared. The lysates were immunoprecipitated with anti-SOCS3 Ab and then immunoblotted with either anti-IkBa Ab (i) or anti-phosphotyrosine Ab (ii). The lysates were also probed with either anti-SOCS3 Ab (iii) or anti-IkBa Ab (iv) to detect the total levels of SOCS3 and IkBa. D, The PMA-differentiated by guest on September 28, 2021 THP-1 macrophages were transiently transfected with either vector control alone (pCDNA) or SOCS3-overexpression plasmid (pCDNA-myc/SOCS3), and after 36 h, cell lysates were prepared. Also, PMA-differentiated THP-1 macrophages were treated with 3 mg/ml rPPE18 for 1 h, and cell lysates were prepared. The cell lysates were either blotted for SOCS3 (top panel) or immunoprecipitated with anti-SOCS3 Ab and then blotted for IkBa (bottom panel). Data are representative of three independent experiments with similar results. also shown to suppress LPS-mediated TNF-a and CD40 expres- The PPE18 protein was found to inhibit LPS-mediated nuclear sion (27, 75). Recently, SOCS3 has also been found to inhibit translocation of NF-kB/rel subunits recruiting the phosphorylated TNFR-associated factor 6 and TGF-b–activated kinase, both of SOCS3 protein to the IkBa–NF-kB/rel complex. Silencing of which are crucial for TLR-driven proinflammatory responses (34). SOCS3 gene expression by a specific siRNA abolished the in- Owing to the importance of SOCS3 protein in the regulation of hibitory effect of PPE18 on NF-kB activity, indicating a role of innate and adaptive immunity (26, 29, 35, 76), it is believed that SOCS3 in the regulation of NF-kB signaling. Phosphorylation of the microorganisms may have evolved strategies to hijack the the IkBa proteins on N-terminal serine residues (serine 32/36) by SOCS3 signaling system also for evasion of the host’s immune IKK-a/b followed by its degradation is a critical process that response. Even though mycobacteria species are suggested to regulates NF-kB activity (56, 57). IKK-b is found to be more trigger SOCS3 expression (25, 31, 32), the pathophysiological role proactive in phosphorylation of IkBa as compared with IKK-a of SOCS3 in tuberculosis is not well understood. In this study, we and appears to be the most dominant kinase in regulating the demonstrate that the PPE18 protein of M. tuberculosis, which is phosphorylation of IkBa and therefore in regulating the NF-kB known to be expressed during infection (77), could also induce activity in our study as also reported by others (58, 80–82). The tyrosine phosphorylation of SOCS3, which was found to be es- most interesting fact uncovered by our study is that a phosphory- sential for suppression of proinflammatory cytokine production lated form of SOCS3 induced by the mycobacterial PPE pro- in LPS-activated macrophages. We have shown earlier that the tein (PPE18) physically interacts with IkBa–NF-kB/rel complex, PPE18 protein specifically interacts with the LRR 11–15 domain which possibly masks the IKK-b substrate residues on IkBa and of TLR2 and activates early induction of IL-10 in macrophages thereby sequesters NF-kB proteins in the cytosol but without (24). Several studies demonstrated that IL-10 enhances SOCS3 affecting IKK-b activity per se. expression (27, 78, 79). Thus, it is possible that IL-10 is involved Although SOCS3 in the presence of rPPE18 could interact with in the increased production of SOCS3 by PPE18 that in turn the IkBa–NF-kB/rel complex, constitutive expression of SOCS3 blocks proinflammatory cytokine induction in LPS-activated mac- protein in the macrophages was not sufficient to interact with the rophages. In addition, PPE18 can also target some other signaling IkBa–NF-kB/rel complex. The apparent difference in binding of pathways to trigger SOCS3 expression and its tyrosine phos- endogenous SOCS3 from SOCS3 induced by rPPE18 was attrib- phorylation. uted to increased tyrosine phosphorylation of SOCS3 induced by 5422 SOCS3 REGULATES NF-kB/rel SIGNALING IN TUBERCULOSIS Downloaded from

FIGURE 8. rPPE18 induces expression and its tyrosine phosphorylation of SOCS3 through activation of p38 MAPK and Src PTKs, respectively. A, The PMA-differentiated THP-1 macrophages were pretreated with 2.5 or 5 mg/ml SB203580 for 1 h followed by incubation with 3 mg/ml rPPE18. After 1 h, http://www.jimmunol.org/ cells were harvested, and SOCS3 mRNA expression was estimated by semiquantitative RT-PCR as described in the Materials and Methods. The mRNA levels were normalized to b-actin mRNA levels. B, Phosphorylation status of Src PTKs in PMA-differentiated THP-1 macrophages on treatment with rPPE18 was monitored by flow cytometry at indicated time points. C, The rPPE18-induced tyrosine phosphorylation of SOCS3 was assessed in PMA- differentiated THP-1 macrophages pretreated for 1 h with PP2 (5 and 10 mM), SU6656 (5 and 10 mM), or Src inhibitor-1 (1 and 5 mM). The cell lysates were immunoprecipitated (IP) with goat anti-SOCS3 Ab, and then immunoblotted (WB) with mouse anti-phosphotyrosine Ab. Data are representative of three independent experiments with similar results. rPPE18. These observations suggest that phosphorylation of ty- during stimulation with LPS, masking the IKK-b substrate site on rosine residues on SOCS3 is essential for its ability to interact IkBa possibly by steric hindrances that prevent IKK-b from by guest on September 28, 2021 with the IkBa–NF-kB/rel complex. Further, we could not detect accessing the serine 32/36 substrate sites on IkBa in the presence an interaction of SOCS3 with the IkBa–NF-kB/rel complex in the of phosphorylated SOCS3. This prevents nuclear translocation of presence of LPS, though LPS is known to increase the levels of NF-kB/rel subunits and induction of proinflammatory cytokine SOCS3, presumably because SOCS3 is not phosphorylated when genes, mainly IL-12 and TNF-a, in activated macrophages. This stimulated with LPS alone (83) (Supplemental Fig. 6). Probably, study also hints to an existence of a novel mechanism of regula- this also explains why SOCS3 does not affect TNF-a secretion tion of NF-kB/rel activity involving SOCS3 in addition to the and NF-kB activity when macrophages are stimulated with LPS classical IKK pathway (48, 56). alone (35, 84). Phosphorylation of SOCS3 at the tyrosine residue There are several reports that have implicated p38 MAPK (s) probably induces a conformational change in the SOCS3, in playing a major role in induction of SOCS3 (32, 65–67). Be- which makes it capable of physically interacting with the IkBa– cause earlier we found that PPE18 interacts with the LRR 11–15 NF-kB/rel complex and sterically prevents IKK-b from access- domain of TLR2 and leads to an early and sustained activation of ing its substrate interface on IkBa. However, the mechanism(s) p38 MAPK, which was critical for IL-10 induction (24), it was by which PPE18 induces tyrosine phosphorylation on SOCS3 reasonable to expect that PPE18 increased the SOCS3 levels in remains to be investigated. Also, further studies are required to THP-1 macrophages via activation of p38 MAPK. Indeed, PPE18- identify the SOCS3 domain that interacts with the IkBa–NF-kB/ mediated activation of SOCS3 was inhibited in the presence of rel complex. SOCS3 is tyrosine-phosphorylated in response to SB203580, a pharmacological inhibitor of p38 MAPK, indicating a variety of stimuli (63, 68, 85), and tyrosine phosphorylation that p38 MAPK is probably involved in the increased induction of of SOCS3 is found to be important for its biological functions. SOCS3 in macrophages treated with PPE18. Using specific Src The principal phosphorylation sites on SOCS3 are residues 204 PTK inhibitors, we provide evidence that Src PTKs, which we and 221 at the C terminus. Studies indicate that tyrosine-phos- found to be active on treatment with PPE18, are involved in the phorylated SOCS3 interacts and inhibits JAK1 phosphorylation tyrosine phosphorylation of SOCS3. Taken together, these results in T cells (63). Similarly, tyrosine-phosphorylated SOCS3 was suggest that SOCS3 phosphorylation is an Src PTK-mediated also shown to interact with and prevent the function of the p120 phenomenon, but the role of other kinases cannot be ruled out. RasGAP (64). Tyrosine-phosphorylated SOCS3 was also found to Therefore, it can be postulated that early activation of p38 MAPK interact with Nck and Crk-L adapter proteins and regulate Nck may provide a pool of SOCS3, which can be rapidly phosphory- activation (62). In this study, we report that a mycobacterial lated by Src PTKs. Taken together, the study points toward a protein can upregulate SOCS3 expression as well as its tyrosine complex regulation of PPE18-induced SOCS3 by p38 MAPK, phosphorylation, and the phosphorylated SOCS3 then interacts and SOCS3 phosphorylation by activating Src PTKs and the N- with the IkBa–NF-kB/rel complex to inhibit phosphorylation of terminal domain of PPE18 was found to be important in mediating IkBa at the serine 32/36 residues by IKK-b in macrophages tyrosine phosphorylation of SOCS3. The Journal of Immunology 5423

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