cIAP1/2−TRAF2−SHP-1−Src−MyD88 Complex Regulates Lipopolysaccharide-Induced IL-27 Production through NF- κB Activation in This information is current as Human Macrophages of September 26, 2021. Aurelia Busca, Yulia Konarski, Niranjala Gajanayaka, Shifawn O'Hara, Jonathan Angel, Maya Kozlowski and Ashok Kumar J Immunol 2018; 200:1593-1606; Prepublished online 22 Downloaded from January 2018; doi: 10.4049/jimmunol.1700199 http://www.jimmunol.org/content/200/5/1593 http://www.jimmunol.org/

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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 © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology cIAP1/2–TRAF2–SHP-1–Src–MyD88 Complex Regulates Lipopolysaccharide-Induced IL-27 Production through NF-kB Activation in Human Macrophages

Aurelia Busca,*,†,1 Yulia Konarski,†,1 Niranjala Gajanayaka,‡ Shifawn O’Hara,† Jonathan Angel,†,x Maya Kozlowski,† and Ashok Kumar*,†,‡

The inhibitors of apoptosis (IAP) , initially described in the context of apoptosis regulation as promoting cell survival, have recently emerged as key regulators of innate immune signaling. As a result, downregulation of IAP via Smac mimetics (SMM) has both survival and immunoregulatory effects. IAPs modulate cytokine production in murine models either as a single agent or in response to LPS. However, the role of SMM and the involvement of IAPs in primary human cells and in particular macrophages

with respect to cytokine production and innate immune responses remain largely unknown. IL-27, a member of the IL-12 cytokine Downloaded from family produced by APCs such as macrophages, has broad immunoregulatory properties in both innate and adaptive immune responses. Herein, we show that cellular IAPs (cIAPs) positively regulate LPS-induced IL-27 production in both primary human monocytes and macrophages. Investigations for the signaling mechanism of cIAPs involvement in IL-27 production in human mac- rophages revealed that LPS-induced IL-27 production is regulated by a novel signaling complex comprising cIAP1/2, TNFR- associated factor 2 (TRAF2), SHP-1, Src, and MyD88 leading to p38, c-Jun N-terminal kinases (JNK) and Akt activation and

NF-kB signaling. In cancer cells, SMM induce the production of cytokines by activating the noncanonical alternate NF-kB http://www.jimmunol.org/ pathway. However, in human macrophages, SMM do not induce the production of TNF-a and other cytokines while inhibiting LPS-induced IL-27 production by inhibiting the classical NF-kB pathway. These signaling pathways may constitute novel ther- apeutic avenues for immune modulation of IL-27 and provide insight into the modulatory immune effects of SMM. The Journal of Immunology, 2018, 200: 1593–1606.

nterleukin-27, a heterodimeric IL-12 family cytokine, is cytokine production in response to TLR2 and TLR4 stimulation and composed of EBV-induced 3 (EBI3) and p28, similar to attenuates IL-10 secretion by human monocytes (1). It inhibits Th1,

I the IL-12 p40 and p35 subunits, respectively. It is secreted Th2, and Th17 immune responses (1–3), prevents the generation of by guest on September 26, 2021 mainly by APCs such as macrophages and dendritic cells (DCs). inducible regulatory T cells (4), and promotes IL-10 synthesis by Initially reported as promoting Th1 differentiation, it exhibits both T cells (5). It displays anti-inflammatory properties by suppressing pro- and anti-inflammatory properties. It augments proinflammatory TNF-a and IL-1b responses in human macrophages (6). It also ex- hibits antitumor activity (7), inhibits HIV replication in CD4+ T cells *Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, and macrophages (8, 9), and has been linked to autoimmune diseases † Ontario K1H 8M5, Canada; Department of Biochemistry, Microbiology and Immu- including Crohn’s disease and experimental autoimmune encephalitis nology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada; ‡Research Insti- tute, Children’s Hospital of Eastern Ontario, University of Ottawa, Ottawa, Ontario (5, 10, 11). High levels of IL-27 have been detected at inflammatory x K1H 5B2, Canada; and Ottawa Hospital Research Institute, University of Ottawa, sites during infection with Mycobacterium tuberculosis (12), Tri- Ottawa, Ontario K1H 8L6, Canada churis muris (13), and Toxoplasma gondii (14). 1 A.B. and Y.K. contributed equally to this work. Recently, transcriptional regulation of p28 and EBI3 subunits ORCIDs: 0000-0001-6350-2030 (N.G.); 0000-0003-3102-4462 (J.A.); 0000-0002- has been studied in murine macrophages and DCs (15, 16). The 3615-8681 (A.K.). adaptive proteins, MyD88 and TIR (Toll/IL-1R) domain–con- Received for publication February 8, 2017. Accepted for publication December 21, 2017. taining adaptor-inducing IFN-b (), regulate IL-27 p28 and This work was supported by grants from the Canadian Institutes of Health EBI3 transcription following LPS stimulation of murine bone Research (HOP 98830 and HOP-107542) to A.K. and by Canadian HIV Cure marrow–derived DCs and macrophages (15–17). TLR-induced Enterprise Team Grant HIG-133050 to A.K. from the Canadian Institutes of Health IL-27 production was also found to be dependent on type I IFN Research in partnership with the Canadian Foundation for AIDS Research and the International AIDS Society. A.B. was supported by an Ontario HIV Treatment responses including IRF-1 and IRF-3 in mouse DCs and human Network studentship. macrophages (15, 18–20). In human DCs, the c-Jun N-terminal Address correspondence and reprint requests to Dr. Ashok Kumar or Dr. Maya kinase (JNK) MAPK regulated IL-27 production following stim- Kozlowski, Research Institute, Children’s Hospital of Eastern Ontario, University ulation with TLR4 ligand (21). The c-Rel, p50, and p65 subunits of Ottawa, 401 Smyth Road, Ottawa, ON K1H 5B2, Canada (A.K.) or Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON of the NF-kB pathway were implicated in IL-27 p28 and EBI3 K1H 8M5, Canada (M.K.). E-mail addresses: [email protected] (A.K.) or expression in LPS-stimulated cells (16). [email protected] (M.K.) Interestingly, the role of upstream signaling molecules such as Abbreviations used in this article: cIAP, cellular IAP; DC, dendritic cell; IAP, tyrosine kinases (PTKs), protein tyrosine phosphatases inhibitor of apoptosis; MDM, monocyte-derived macrophage; NIK, NF-kB–inducing kinase; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; siRNA, (PTPs), and inhibitors of apoptosis (IAPs) in IL-27 regulation small interfering RNA; SMM, Smac mimetic; TRAF, TNFR-associated factor; TRIF, remains unknown. PTKs such as Src family kinases were impli- TIR domain–containing adaptor-inducing IFN-b. cated in TNF-a and IL-6 production (22). The activity of the Src Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 kinases is tightly regulated by reversible tyrosine phosphorylation www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700199 1594 cIAP1/2–TRAF2–SHP-1–Src–MyD88 COMPLEX AND IL-27 REGULATION

(23). Sequestration of the Src tyrosine phosphorylated tail by its 10% FBS, followed by the addition of standards and sample supernatants SH2 domain maintains an inactive state of the kinase. Therefore, it for another 24 h. On the third day biotinylated secondary mouse mono- requires a PTP, such as SHP-1, to activate it and promote down- clonal anti-human Abs were added, followed by streptavidin-HRP. Visu- alization was carried out using 3,39,5,59-tetramethylbenzibidine one stream phosphorylation (23, 24). SHP-1 regulates several tyrosine component HRP microwell substrate solution and 450 nm liquid stop so- phosphorylated molecules in hematopoietic cells and plays a role lution for 3,39,5,59-tetramethylbenzibidine microwell substrates (BioFX in LPS-induced IL-12 p40 (25) and IL-6 production in LPS- Laboratories, Owings Mills, MD). Absorbance was read using Bio-Rad stimulated bone marrow–derived murine macrophages (26). iMark microplate reader and data were processed using Microplate Manager 6 software. Initially described in the context of apoptosis regulation as promoting cell survival, the IAP proteins have recently emerged as Confocal laser scanning fluorescence microscopy key regulators of innate immune signaling that promote inflam- Monocytes were initially adhered and differentiated into MDMs as de- mation (27). Macrophages of cellular IAP2 (cIAP2) null mice scribed above on round glass coverslips in 12-well plates. After LPS exhibited an impaired inflammatory response to LPS, and were treatment cells were fixed in 4% paraformaldehyde for 15 min, rinsed in resistant to endotoxic shock (28). IAPs also play a role in innate PBS, and then permeabilized with 0.1% Triton X-100 for another 10 min. immune responses against infections with Chlamydia pneumoniae The coverslips were incubated with anti–SHP-1, anti-Src, anti-MyD88, or anti-cIAP2 rabbit primary Abs ( Technology, Danvers, MA) (29) and Listeria monocytogenes (30). IAPs associate with TLR4 at 4˚C overnight. On the second day coverslips were rinsed with 5% BSA signaling complexes to serve as ubiquitin ligases that promote in PBS and incubated in the same buffer with secondary Abs for 1 h at degradation of adaptor proteins such as TNFR-associated factor 3 room temperature: Alexa Fluor 680 donkey anti-rabbit IgG or Alexa Fluor (TRAF3) (31). cIAP1 and cIAP2 associate with the TNF-a re- 350 donkey anti-rabbit IgG (Molecular Probes, Burlington, ON, Canada). Coverslips were mounted on microscopy slides using ProLong Gold ceptor complex I to mediate TNF-a–induced activation of the antifade mounting media with DAPI (Invitrogen) and examined with a Downloaded from classical NF-kB pathway (32) and at the same time inhibit the Zeiss LSM 510 Meta confocal microscope using a 488 nm (green), a 633 nm alternative NF-kB pathway by promoting constant degradation of HeNe (red), or a 405 nm (blue) laser. Fluorescent images were acquired with NF-kB–inducing kinase (NIK), the regulatory kinase of non- ZEN 2009 software and analyzed with ImageJ software. canonical NF-kB signaling (33). However, the involvement of Western blot analysis IAPs in cytokine production by primary cells and particularly Total cell proteins obtained after lysis of cell pellets were subjected to SDS- human macrophages is poorly understood. Herein, we show for http://www.jimmunol.org/ PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad the first time, to our knowledge, that IAPs associate with Src, Laboratories, Hercules, CA) as described earlier (34, 35). Membranes were SHP-1, MyD88, and TRAF1/2 to form a novel upstream signaling probed with Abs against cIAP1, cIAP2, XIAP, TRAF1, TRAF2, TRAF6, complex that regulates LPS-induced IL-27 production through the MyD88, and RIP-1 (all from Cell Signaling Technology). Anti-p100/p52 activation of downstream p38 and JNK MAPKs, PI3K, and NF-kB Ab was from Sigma-Aldrich. For SHP-1 we used a mouse anti–SHP-1 Ab generated in the laboratory. For experiments involving signaling pathways, in human monocyte-derived macrophages (MDMs). Abs against IkBa, p-Akt, Akt, p-p38, p38 (all from Cell Signaling), p-JNK, and JNK (both from Santa Cruz Biotechnology, Santa Cruz, CA) were used. Membranes were probed with primary Abs at 4˚C overnight, Materials and Methods followed by goat secondary Abs conjugated to HRP (Bio-Rad). To control

Cells and reagents for total protein loading, membranes were stripped of the primary Abs and by guest on September 26, 2021 reprobed with anti-GAPDH (Sigma-Aldrich) or anti–b-actin (Cell Sig- MDMs were obtained from primary monocytes isolated by the adherence naling) Ab. Immunoblots were visualized using the Amersham ECL method as described earlier (34–36). Blood was obtained from healthy Western blotting detection system. The images were obtained with the volunteers according to a protocol approved by the Ethics Review Com- ChemiGenius Bio-imaging system and the GeneSnap software (both from mittee of The Ottawa General Hospital and the Children’s Hospital of Syngene). Eastern Ontario, Ottawa. Briefly, human PBMCs isolated by Ficoll Paque density centrifugation (GE Healthcare Life Sciences, Buckinghamshire, Transfection with small interfering RNA U.K.) were resuspended in serum-free media (5 3 106/ml) and plated in 12-well polystyrene plates (Becton Dickinson, Franklin Lakes, NJ). After All specific small interfering RNAs (siRNAs) were purchased from Santa being allowed to adhere for 3 h, nonadherent cells were washed off and Cruz Biotechnology. MDMs were transfected with RIP-1 siRNA, TRAF1/2 adherent cells were cultured for 6 d in IMDM supplemented with 10% FBS siRNA, cIAP1/2 siRNA, SHP-1 siRNA, Src siRNA, Akt siRNA, p38 and 10 ng/ml M-CSF (R&D Systems, Minneapolis, MN). Media con- siRNA, JNK1 and 2 siRNA (Santa Cruz Biotechnology) or control siRNA taining M-CSF was replenished every 2 d. MDMs thus obtained were 98% (Qiagen, Venlo, the Netherlands) using TransIT-TKO transfection reagent CD14+. Primary monocytes were obtained from PBMCs by using Miltenyi (Mirus Bio, Madison, WI), as described earlier (34–36). Briefly, 40 pmol monocyte negative selection kit II (Miltenyi Biotech, Auburn, CA) as per siRNA was mixed with 4 ml transfection reagent in 100 ml of serum-free the manufacturer’s instructions. Negatively selected monocytes were pu- media to allow formation of RNA complexes. After 30 min, the siRNA rified using Automacs sorting. mixtures were added to cells. After 5 h, the media of transfected cells was LPS was obtained from Escherichia coli O111:B4 (Sigma, St. Louis, changed to complete media. The following day after transfection, cells MO), dissolved in complete medium, aliquoted, and stored at 280˚C be- were treated with LPS for another 24 h, then supernatants and cell pellets fore use. Second mitochondria-derived activator of caspase (Smac) mi- were collected for cytokine evaluation and protein extraction respectively. metics (SMM) LN730 and SM164 were a generous gift from Dr. Korneluck (Children’s Hospital of Eastern Ontario Research Institute, RT-PCR Apoptosis Research Centre). Chemical inhibitors LY294002 (PI3K/Akt 6 MDMs (1 3 10 cells per well) were stimulated with LPS for 4 h. RT-PCR inhibitor), sodium stibogluconate (SHP-1 inhibitor), SU6656 (Src inhibi- for IL-27 subunits was performed as described earlier (37). RNA was tor), SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), and CAPE extracted using RNeasy kit (Qiagen, Mississauga, ON, Canada) according k (NF- B inhibitor) were purchased from Calbiochem and used to treat to the manufacturer’s protocol and as described earlier (34–36). For RT- MDMs for 2 h before LPS stimulation. PCR, a master mix was prepared with 7.5 ml103 reverse transcriptase Cytokine measurement by ELISA buffer, 3 ml dNTP, 7.5 ml random primers, 3.75 ml reverse transcriptase, and 53.25 ml double distilled H2O. Twenty-five microliters of master mix IL-27 measurement in culture supernatants was performed using IL-27 was added to each sample to 50 ng/ml purified RNA and RT-PCR was ELISA DuoSet kit purchased from R&D Systems (catalog number carried out for 2 h in a GeneAmp PCR System 2700 amplifier (Applied DY2526) following the manufacturer’s instructions and as described ear- Biosystems, Carlsbad, CA) to yield cDNA. The PCR program was as lier (37). Briefly, microplates were coated overnight at 4˚C in ELISA follows: initial incubation for 2 min at 50˚C and 10 min at 95˚C, and cycles coating buffer (eBioscience, San Diego, CA) with 0.4 mg/ml IL-27 capture of denaturation at 95˚C for 15 s were followed by annealing and elongation Ab (R&D Systems). For measurement of TNF-a secretion, 0.2 mg/ml for 2 min at 60˚C. Forty to fifty cycles were applied. cDNA was then used TNF-a capture Ab (Invitrogen, Burlington, ON, Canada) was used with in real-time PCR reactions. For this, samples were prepared with 12.5 ml a similar protocol. The next day plates were washed, blocked for 2 h with TaqMan DNA polymerases, 1.25 ml primer pairs (for each of the cytokine The Journal of Immunology 1595 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. SMM inhibit LPS-induced IL-27 production in MDMs and primary human monocytes. (A) MDMs were treated with the indicated concentrations of SMM LN730 or SM164 for 24 h. Cells were collected and analyzed for IAP protein expression by Western blotting. (B and D) MDMs were treated with 50 nM of SMM LN730 (B)orSM164(D) for 24 h, followed by another 24 h of LPS 100 ng/ml stimulation. (C and E) Primary monocytes were treated concomitantly with the indicated SMM and LPS (1 mg/ml) for 24 h. Supernatants were evaluated for IL-27 expression by ELISA as described in Materials and Methods.Eachsymbolon the dot plots represents an individual donor. Horizontal lines indicate mean values. Bar graphs show the mean 6 SD of percentage IL-27 secretion relative to maximal concentration induced by LPS, which was considered 100%. n =3–9.*p , 0.05. (F and G) MDMs were transfected with the indicated concentrations of cIAP1 and cIAP2 siRNA or control siRNA as described in Materials and Methods. After 24 h, cells were stimulated (Figure legend continues) 1596 cIAP1/2–TRAF2–SHP-1–Src–MyD88 COMPLEX AND IL-27 REGULATION

subunits), and 8.75 ml double distilled H2O. Real-time PCR was carried kinase, is involved in TNF-R1 signaling and IAP-mediated out for 2 h in a 7500 Real-Time PCR System. The expression levels of the apoptosis in cancer cells, p38 MAPK activation in response to transcripts were shown as the ratio compared with b-actin by calculation of TNF-a, and NF-kB activation via TLR3 and TLR4 (42–45). To cycle threshold values in amplification plots (Applied Biosystems). Primer pairs (Applied Biosystems, U.K.) were as follows: IL-27 EBI3 primer: understand the involvement of IAPs, we investigated if RIP-1 59-AGCAGCAGCCTCCTAGCCT-39,59-ACGCCTTCCGGAGGGTC-39; was involved in SMM-mediated inhibition of LPS-induced IL-27 IL-27 p28 primer: 59-GGCCAGGYGACAGGAGACC-39,59-CAGCTTG- production. RIP-1 levels were increased following LPS stimu- TACCAGAAGCAAGGG-39. lation of MDMs and this effect was inhibited following SMM Immunoprecipitation pretreatment (Fig. 2A). The role of RIP-1 in SMM-mediated inhibition of LPS-induced IL-27 production was determined by MDMs were treated with SMM LN730 for 24 h, followed by LPS (100 ng/ml) for another 24 h. Cell lysates were collected by treating cell pellets with using RIP-1 siRNA. Despite successful knockdown of RIP-1 lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% (Fig. 2B), LPS-induced IL-27 production remained unchanged Triton X-100, 1.5 mM MgCl2, 100 mM NaF, 100 mM sodium orthova- (Fig. 2C). These results suggest that SMM-mediated inhibi- nadate, and 1 mM EGTA pH 7.7) for 1 h on ice. Two hundred micrograms tion of LPS-induced IL-27 production does not involve RIP-1 m of protein for each sample was mixed with 20 l of Protein A Sepharose induction. beads (Invitrogen) and specific coimmunoprecipitation Abs (either anti- MyD88, anti-TRAF1, or anti-TRAF2 Abs, all from Cell Signaling) and TRAF2 and MyD88 regulate LPS-induced IL-27 production. To the mixture was incubated overnight at 4˚C. Following overnight incuba- further dissect the mechanism of SMM inhibition of LPS signaling, tion, samples were centrifuged and the beads with attached Abs and we investigated the effect of SMM on proteins involved in TLR4 coimmunoprecipitated proteins were collected. The beads were resus- pended in loading dye, boiled for 5 min to release the coimmunoprecipi- signaling. LPS binding to TLR4 triggers two distinct signaling tated protein–Ab complexes, then separated by SDS-PAGE and evaluated cascades according to different adaptor molecules recruited to the Downloaded from by Western blotting as described above. , MyD88 and TRIF, that culminate with cytokine pro- Statistical analyses duction and IFN responses, respectively (46). TRAFs are molecules involved in multiple transduction path- Data was plotted using Windows Excel 2010 (Microsoft) and GraphPad ways, such as TLR and TNFR signaling. Of the six TRAF mol- Prism 5. To minimize variability between donors in their responsiveness to ecules, TRAF6 is implicated in TLR4 signaling downstream of LPS, data were calculated relative to LPS response, which was considered 100%. Statistical analysis of multiple groups was performed using ANOVA, MyD88, where it serves as ubiquitin ligase that activates IkB ki- http://www.jimmunol.org/ followed by Tukey post hoc test. Two groups were compared using Student t nase and classical NF-kB signaling (47). TRAF2 is mainly in- test. A p value ,0.05 was considered significant. Unless otherwise spec- volved in TNF signaling, whereas the role of TRAF1 is to recruit 6 ified, plotted data represent the mean SD of at least three experiments. cIAP1 and 2 to TRAF2, thereby forming a cIAP1/2-TRAF1/2 heterocomplex (48). Results Initially, we evaluated if SMM affect TRAF1, 2, 6, and cIAPs regulate LPS-induced IL-27 production in human MyD88 expression levels. LN730 treatment inhibited LPS- macrophages induced TRAF1, TRAF2, and MyD88, with no effect on IAP antagonists (SMM) have been evaluated in clinical trials for TRAF6 (Fig. 2D) suggesting that TRAF1, TRAF2, and MyD88 cancer treatment for their ability to promote autoubiquitination and may be involved in SMM-mediated inhibition of LPS-induced by guest on September 26, 2021 proteosomal degradation of IAPs leading to TNF-a–induced ap- IL-27 secretion. To determine their specific involvement, we optosis in cancer cells (38–41). To understand the role of IAPs on used siRNAs for TRAF1, TRAF2, and MyD88, which induced cytokine production by primary human macrophages, we inves- specific knockdown of their targets (Fig. 2E, 2F, left panels). tigated the impact of SMM (LN730 and SM164) on LPS-induced IL-27 production was significantly inhibited in response to LPS IL-27 production in TLR4-stimulated human MDMs. LN730 in cells transfected with TRAF2 and MyD88 siRNA compared significantly decreased basal levels of cIAP1, cIAP2, and XIAP, with cells transfected with control siRNA (Fig. 2E, 2F, right whereas SM164 affected only cIAP1 and cIAP2 (Fig. 1A). LN730 panels), suggesting that MyD88 and TRAF2 are the main significantly inhibited IL-27 secretion in response to LPS in adaptor molecules in LPS signaling that are responsible for MDMs (Fig. 1B) and primary monocytes (Fig. 1C). Similar results IL-27 production. showing inhibition of LPS-induced IL-27 production following SHP-1 and Src regulate LPS-induced secretion of IL-27 in SM164 treatment of MDMs (Fig. 1D) and primary monocytes human MDMs (Fig. 1E) were observed suggesting that this effect is not limited to macrophages or to one SMM. The involvement of cIAP1/2 was The PTK Src has been shown to regulate the production of confirmed using specific siRNAs for cIAP1 and cIAP2, which proinflammatory cytokines such as IL-6 (22, 26). Src requires a effectively inhibited cIAP1/2 expression without affecting XIAP protein phosphatase SHP-1 to form a signaling tandem that expression (Fig. 1F). There was a significant reduction of LPS- dephosphorylates Src at the inhibitory tyrosine site (49). induced IL-27 production in MDMs transfected with cIAP1 and Therefore, we determined the involvement of Src and SHP-1 in cIAP2 siRNAs compared with those transfected with control the regulation of LPS-induced IL-27 production. Treatment of siRNA (Fig. 1G). MDMs with either sodium stibogluconate (SS, SHP-1 inhibi- tor) (Fig. 3A) or SU6656 (SU, Src inhibitor) (Fig. 4A) resulted Role of IAP-associated signaling molecules in LPS-induced in significant suppression of IL-27 protein. Similar results IL-27 production were obtained with IL-27 subunits at the mRNA level, with RIP-1 does not regulate LPS-induced IL-27 production. The both IL-27 p28 mRNA (Figs. 3B, 4B) and EBI3 mRNA receptor-interacting protein 1 (RIP-1), a containing (Figs. 3C, 4C) showing suppression following treatment with

with LPS (100 ng/ml) for another 24 h, following which cell pellets were collected for evaluation of antiapoptotic proteins by Western immunoblotting(F), and supernatants were collected for IL-27 measurement by ELISA (G). Bar graph shows the mean 6 SD of percentage IL-27 secretion relative to maximal concentration induced by LPS, which was considered 100%. n =4.*p , 0.05. The Journal of Immunology 1597 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. TRAF2 and MyD88 regulate LPS-induced IL-27 production in MDMs. (A) MDMs were treated with LPS 100 ng/ml in the presence or absence of LN730 pretreatment for 24 h at the indicated concentrations. Whole protein lysates were evaluated for RIP-1 expression by Western blotting. GAPDH was used as a loading control and cIAP2 was used as a positive control for LN730 activity. (B and C) MDMs were transfected with control or RIP-1 siRNA for 24 h as described in Materials and Methods followed by LPS stimulation for another 24 h. The total cell lysates were evaluated for RIP-1 expression by Western blotting (B). The supernatants were analyzed for IL-27 production by ELISA (C). The bar graph shows the mean 6 SD of percentage IL-27 secretion relative to maximal concentration induced by LPS, which was considered 100%. n =4.(D) MDMs were treated with LPS in the presence or absence of LN730. The cell lysates were evaluated for TRAF1, TRAF2, TRAF6, and MyD88 expression by Western blotting with GAPDH as a loading control. The results are representative of three different donors. Right panel shows mean of relative densities of TRAF1, TRAF2, TRAF6, and MyD88. (E and F)TheMDMs were transfected with control, TRAF1, or TRAF2 siRNA (E), or MyD88 siRNA (F) for 24 h followed by stimulation with LPS for another 24 h. The total cell lysates were evaluated for TRAF1, TRAF2, and MyD88 expression by Western blotting (left panels) and the supernatants were assayed for IL-27 production by ELISA (right panels). The bar graphs show the mean 6 SD of percentage IL-27 secretion from three different donors, relative to the maximal concentration induced by LPS in control siRNA–treated cells, which was considered 100%. *p , 0.05. 1598 cIAP1/2–TRAF2–SHP-1–Src–MyD88 COMPLEX AND IL-27 REGULATION Downloaded from

FIGURE 3. SHP-1 positively regulates LPS-induced IL-27 production in MDMs. MDMs were treated with indicated concentrations of sodium stibo- http://www.jimmunol.org/ gluconate (SS, inhibitor for SHP-1) for 2 h (A–C) or transfected with SHP-1 siRNA for 5 h (D–F), followed by LPS stimulation for 24 h for IL-27 protein measurement (A and D) or for 4 h for RT-PCR analysis (B, C, E, and F). Subsequently, samples were collected and IL-27 was measured by ELISA in the supernatants (A and D). EBI3 and p28 transcripts were measured by RT-PCR (B, C, E, and F) and quantified relative to LPS, which was considered as 100%. Bar graphs in (B), (C), (E), and (F) show mean 6 SD of IL-27 p28 or EBI3 mRNA transcripts. n =4.*p , 0.05. (G) MDMs were transfected with either control or SHP-1 siRNA followed by LPS stimulation. Cell pellets were analyzed by Western immunoblotting for SHP-1. SHP-1 or Src inhibitor respectively. These results were con- target proteins (Figs. 3G, 4G). There was a significantly de- firmed using siRNAs specific to SHP-1 and Src, which were creased production of IL-27 protein and IL-27 p28 and EBI3

efficient in downregulating the basal level of their respective mRNAs in cells transfected with SHP-1 (Fig. 3D–F) and Src by guest on September 26, 2021

FIGURE 4. Src positively regulates LPS-induced IL-27 production in MDMs. MDMs were treated with indicated concentrations of SU6656 (SU, in- hibitor for Src) for 2 h (A–C) or transfected with Src siRNA for 5 h (D–F), followed by LPS stimulation for 24 h for IL-27 protein measurement (A and D)or for 4 h for RT-PCR analysis (B, C, E, and F). Subsequently, supernatants were collected and IL-27 was measured by ELISA (A and D). EBI3 and p28 transcripts were measured by RT-PCR (B, C, E, and F) and quantified relative to LPS, which was considered as 100%. Bar graphs in (B), (C), (E), and (F) show mean 6 SD of IL-27 p28 or EBI3 mRNA transcripts. n =4.*p , 0.05. (G) MDMs were transfected with either control or Src siRNA followed by LPS stimulation. Cell pellets were analyzed by Western immunoblotting for Src. The Journal of Immunology 1599 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. SHP-1, Src, MyD88, and cIAP2 colocalize upon LPS stimulation. MDMs were either left untreated or stimulated with LPS for 15 min following which cells were stained with Abs specific for SHP-1 and Src (A), SHP-1 and MyD88 (B), Src and MyD88 (C), or SHP-1 and cIAP2 (D). The cells were analyzed by immunofluorescence as described in Materials and Methods. Images show one representative experiment, from three similar results obtained with cells from three different donors. Original magnification 3630.

(Fig. 4D–F) siRNAs as compared with those transfected with human macrophages. Therefore, it was of interest to determine if control siRNAs. SHP-1/Src signaling and TLR4/MyD88 signaling are inter- connected or function as separate, parallel pathways. We show by cIAP1/2, TRAF1/2, SHP-1, Src, and MyD88 form a signaling immunofluorescence that SHP-1 and Src colocalize following LPS complex in MDMs stimulation in human macrophages (Fig. 5A). Similarly, SHP-1 The above results show that cIAP1/2, PTP SHP-1, PTK Src, (Fig. 5B) and Src (Fig. 5C) were shown to coimmunofluoresce TRAF2, and MyD88 regulate LPS-induced IL-27 production in with MyD88 after LPS stimulation. Furthermore, cIAP2 and SHP-1 1600 cIAP1/2–TRAF2–SHP-1–Src–MyD88 COMPLEX AND IL-27 REGULATION Downloaded from http://www.jimmunol.org/

FIGURE 6. cIAP1/2, TRAF1/2, SHP-1, Src, and MyD88 form a signaling complex in MDMs. MDMs were treated with LPS 100 ng/ml in the presence or absence of LN for 24 h. Cell pellets were used to extract protein and 200 mg of each sample was used with anti-MyD88 (A), anti-TRAF1 (B), anti-TRAF2 (C), or anti-Src (D) Abs as capture Abs to coimmunoprecipitate proteins that would bind to their specific targets, as described in Materials and Methods. The protein–Ab complexes were immunoblotted and membranes were probed with the indicated Abs. Cell lysates without capture Ab or Sepharose beads

were used as controls in the first lane for correct location of the band of interest. Sepharose beads with capture Ab and no protein were used as negative by guest on September 26, 2021 controls in the second lane. Results are representative of three similar experiments, obtained with cells from different donors. colocalized after LPS stimulation (Fig. 5D) suggesting that cIAP2 is This interaction was abolished following SMM pretreatment also a part of the SHP-1–Src–MyD88 signaling complex. (Fig. 6D, lanes 5 and 6). Overall, these results suggest that an To determine that colocalization observed with immunofluores- upstream signaling complex consisting of cIAP1/2–TRAF1/2– cence is due to physical interaction of these molecules, we performed SHP-1–Src–MyD88 regulates LPS-induced IL-27 production in coimmunoprecipitation experiments. Using anti-MyD88 capture Ab, MDMs. results show that cIAP1/2, SHP-1, and Src interacted with MyD88 in media and LPS-treated cells (Fig. 6A, lanes 3 and 4). As expected, cIAP-mediated MAPKs and Akt activation contribute to cIAP1/2 bands were not visible in cells pretreated with SMM before LPS-induced IL-27 production LPS stimulation (Fig. 6A, lanes 5 and 6). However, SMM did not TLR4 signaling in response to LPS leads to MAPKs and NF-kB affect SHP-1 or Src binding to MyD88 (Fig. 6A, lanes 5 and 6). activation to promote cytokine synthesis (50). cIAPs are also These results confirmed that cIAP1/2, SHP-1, Src, and MyD88 in- required for MAPKs activation in response to LPS stimulation teract to form a signaling complex. (51). Moreover, MAPKs can also be activated downstream of We next determined if TRAF1/2 were also a part of the Src/SHP-1 kinases (52). To determine if IAPs influence signaling complex that includes cIAP1/2. TRAF1/2, the binding MAPKs and PI3K/Akt activation leading to LPS-induced IL-27 partners of cIAP1/2, could not be evaluated on these Western production, we first determined the requirement for MAPKs blot membranes, as TRAFs 50 kDa bands were masked by the and Akt in the regulation of IL-27 production by using inhib- capture Ab H chain band. Therefore, we used either TRAF1 or itors specific for JNK (SP600125), p38 (SB203580), and Akt TRAF2 as capture Abs in subsequent coimmunoprecipitation (LY294002). Blocking of JNK, p38, and Akt significantly experiments. Results show that cIAP1/2, SHP-1, Src, and inhibited LPS-induced IL-27 production (Fig. 7A–C, left pan- MyD88bindtoTRAF1(Fig.6B,lanes3and4)andTRAF2 els). Interestingly, JNK MAPK had a divergent effect on the (Fig. 6C, lanes 3 and 4) in media and LPS-treated cells. Similar two IL-27 subunits at mRNA levels, as it positively regulated to the MyD88 results, there was decreased binding of cIAP1/2 LPS-induced IL-27 p28 mRNA without any effect on EBI3 to TRAF1 (Fig. 6B, lanes 5 and 6) and TRAF2 (Fig. 6C, lanes 5 mRNA (Fig. 7A). Similarly, p38 MAPK positively regulated and 6) when cells were treated with SMM. Moreover, SMM LPS-induced IL-27 EBI3 mRNA, with no effect on p28 mRNA pretreatment did not influence TRAF1/2 binding to SHP-1, Src, (Fig. 7B). These results suggest that one of the subunits rep- and MyD88. These results were confirmed using Src as the resents the limiting factor in protein secretion. PI3K regulated capture Ab, which showed cIAP2 interactions with the Src- both p28 and EBI3 mRNA (Fig. 7C). The requirement for JNK, SHP-1 complex upon LPS induction (Fig. 6D, lanes 3 and 4). p38, and Akt signaling was confirmed by using their respective The Journal of Immunology 1601 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. p38, JNK, and Akt regulate LPS-induced IL-27 production in MDMs. (A–C) MDMs were treated for 2 h with the indicated concentrations of pharmacological inhibitors SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), and LY294002 (PI3K/Akt inhibitor) before LPS stimulation. After 24 h of LPS treatment, supernatants were collected and evaluated for IL-27 by ELISA (left panels), whereas EBI3 (right panels) and p28 (middle panel) transcripts were measured by quantitative RT-PCR as described in Materials and Methods. Bar graphs show the mean 6 SD of percentage IL-27 secretion or IL-27 p28 and EBI3 mRNA transcripts relative to maximal concentration induced by LPS, which was considered 100%. n = 3–5. *p , 0.05. (D) MDMs were transfected with JNK1/2 siRNA, p38 siRNA, Akt siRNA, or control siRNA for 24 h before adding LPS for another 24 h. Next, the supernatants were assayed for IL-27 production by ELISA (upper panels), whereas whole cell lysates were evaluated for their respective phosphorylated proteins, namely JNK (left panel), p38 MAPK (middle panel), and Akt (right panel), by Western immunoblotting (lower panels). Bar graphs show the mean 6 SD of percentage IL-27 secretion relative to maximal concentration induced by LPS, which was considered 100%. n = 3–5. *p , 0.05. specific siRNAs, which significantly inhibited LPS-induced ylation of JNK, p38, and Akt (Fig. 8A, 8B), suggesting that IL-27 production compared with control siRNA treated cells SMM inhibit LPS-induced IL-27 production through the inhi- (Fig.7D).Moreover,SMMinhibited LPS-induced phosphor- bition of p38 and JNK MAPKs and PI3K/Akt signaling. 1602 cIAP1/2–TRAF2–SHP-1–Src–MyD88 COMPLEX AND IL-27 REGULATION

Noncanonical regulation of NF-kB depends on NIK, which promotes partial processing of the p100 subunit to its p52 mature form (54). In resting cells, this pathway is subdued via contin- uous NIK degradation performed by cIAP1/2 (33). SMM treat- ment as a single agent leads to IAP inhibition resulting in NIK accumulation and subsequent activation of noncanonical NF-kB pathway in cancer cells (38, 39). The effect of SMM on alter- native NF-kB signaling in human macrophages is not known. As a single agent, in contrast to cancer cells (38, 39), SMM did not impact p100 processing in macrophages. However, SMM aug- mented LPS-induced p100 processing to p52 (Fig. 9B), indi- cating that SMM promote alternative NF-kB signaling, in contrast to the inhibitory effect on the classical pathway fol- lowing LPS stimulation. To determine if the SMM effect on secretion of IL-27 could be attributed to altered NF-kB signaling, we employed the chemical inhibitor CAPE to block NF-kB pathway. Inhibition of NF-kB by CAPE significantly reduced LPS-induced IL-27 production

(Fig. 9C). Given the requirement for NF-kB in regulating IL-27 Downloaded from and the inhibitory effect of SMM on the classical pathway, these results suggest that IAPs regulate IL-27 induction following TLR4 stimulation through the classical NF-kB pathway. More- over, activation of a noncanonical alternate pathway by SMM in LPS-activated macrophages does not impact IL-27 production

(Fig. 10). http://www.jimmunol.org/

Discussion SMM compounds have been actively studied for their apoptosis- inducing abilities in cancer cells (38, 39). We and others have shown that cIAP ablation on its own does not influence survival of primary human macrophages (34, 35, 55) and murine B cells FIGURE 8. LPS-induced p38, JNK, and Akt activation is dependent on (33). However, the role of IAPs in regulating immune responses IAPs. MDMs were treated with SMM LN for 24 h before stimulation with in primary cells is poorly understood. One report from mouse

LPS for the indicated times. Cells were collected and evaluated for by guest on September 26, 2021 studies indicates that cIAPs mediate the production of proin- phosphorylation status of JNK (A), p38 (B), and Akt (C) by Western blotting. GAPDH or actin was used as a loading control. Results shown are flammatory cytokines in response to LPS, without affecting IFN representative of at least three different donors. responses. As a result, cIAP ablation using SMM resulted in decreased IL-6, TNF-a, and IL-12 production, with no effect on IL-10 (31). However, the effect of SMM on cytokine production in primary human cells remains unknown. SMM have also been k cIAP-mediated classical NF- B signaling contributes to LPS- used in phase 1 and 2 clinical trials as therapeutic options in induced IL-27 production various cancers (40, 41). Therefore, it is imperative to under- Because TLR4 signaling culminates with NF-kB activation, we stand the impact of SMM on immune signaling in primary evaluated if cIAP1/2 regulate LPS-induced IL-27 production human cells. Herein, we show that cIAPs positively regulate through NF-kB signaling in MDMs. cIAP ablation by SMM as a LPS-induced IL-27 production in human macrophages by a single agent has been shown to promote classical pathway of novel signaling complex comprising cIAP1/2, TRAF2, SHP-1, NF-kB activation in cancer cells because of increased TNF-a Src, and MyD88 leading to p38, JNK, and Akt activation and secretion, which promotes degradation of IkBa protein and NF-kB signaling. subsequent release of p65 and p50 subunits of NF-kB (38, 39). LPS binding to TLR4 recruits two adaptor molecules with Toll/ To determine if SMM inhibit classical NF-kB activation in hu- IL-1 receptor (TIR) domains, MyD88 and TRIF. MyD88 recruits man macrophages, we measured the level of IkBa protein as an IRAK1/4, followed by TRAF6, which ultimately mediates IkB indirect indicator of the classical NF-kB activity. In contrast to kinase complex activation leading to secretion of inflammatory previous results obtained with cancer cells (38, 39), SMM alone cytokines (56). SMM bind specifically to cIAP1 and 2 (45) and did not affect IkBa expression in human macrophages. As ex- cause their degradation without affecting IAP-associated pro- pected, LPS stimulation decreased IkBa levels, indicative of NF- teins such as TRAFs (39). TRAF1/2 bind cIAP1/2 to form a kB activation. However, LPS-induced degradation of IkBa was heterocomplex with key functions in TNFR signaling (57), attenuated in MDMs pretreated withSMM(Fig.9A),suggesting whereas TRAF6 contributes to MyD88-dependent TLR4 sig- that LPS-induced NF-kB activation is dependent on cIAPs in naling (58). Our results show that SMM treatment inhibited LPS- human macrophages. In cancer cells SMM induce cell death by induced TRAF1 and 2, with no effect on TRAF6. However, only promoting the production of TNF-a, a downstream target of TRAF2 was required for IL-27 production. The mechanism re- classical NF-kB pathway with positive feedback effect on NF- sponsible for IL-27 downregulation by TRAF2 and not by kB activation (38, 39, 45, 51, 53). However, in human macro- TRAF1 is not clear. There is a redundancy in the function and phages, we show that SMM do not alter LPS-induced production structure of TRAF1 and TRAF2. Although both TRAF1 and of TNF-a (Fig. 9B), suggesting that this mechanism is not in- TRAF2 can bind cIAP1/2, TRAF1 and TRAF2 preferentially form volved in regulating classical NF-kB pathway. 1:2 trimers that bind with higher affinity to cIAP2 compared with The Journal of Immunology 1603 Downloaded from http://www.jimmunol.org/

FIGURE 9. Involvement of classical NF-kB pathway in IAP-mediated regulation of LPS-induced IL-27 production in MDMs. (A and B) MDMs were treated with SMM (LN730 and SM164) for 24 h, followed by LPS (100 ng/ml) stimulation for 30 min (A)or24h(B). Cells were collected and evaluated for the expression of IkBa (A) and p100 to p52 processing (B) by Western immunoblotting. Results shown are representative of at least three different donors. (C) MDMs from different donors were treated with 50 nM of SMM LN730 for 24 h, followed by another 24 h of LPS 100 ng/ml stimulation. Supernatants were collected and evaluated for TNF-a expression by ELISA as described in Materials and Methods. Each symbol on the dot plot represents an individual donor. Horizontal lines indicate mean values. (D) MDMs were treated for 2 h with the indicated concentrations of CAPE (NF-kB inhibitor), followed by another 24 h of LPS stimulation. Supernatants were collected and evaluated for IL-27 levels by ELISA. Bar graph shows the mean 6 SD of percentage IL-27 secretion relative to maximal concentration induced by LPS, which was considered 100%. n =3.*p , 0.05. by guest on September 26, 2021

TRAF2 alone, indicating the importance of oligomerization for we also show that SHP-1 regulates IL-27 production downstream TRAF1/2 function (59). It is possible that when TRAF1 levels are of TLR4 in human macrophages. reduced, TRAF2 is still able to bind the signaling complex and PTKs such as Src represent targets for SHP-1 activity (68). promote IL-27 production. Secondly, the RING domain and the Under basal conditions, Src is present in a restrictive state, where cIAP1/2-interacting motif within TRAF2 are required to regulate Tyr527 in the C terminus is phosphorylated and binds with the downstream (NIK) stability, mediate activation of the canonical NF- Src SH2 domain (24). This molecular interaction sequesters the kB pathway, and suppress constitutive noncanonical NF-kBacti- catalytic domain and holds the kinase in an inactive state and a PTP, vation (57, 60). Interestingly, RING domain is absent in TRAF1 such as SHP-1, can activate it to promote downstream phosphory- (61–63). This may explain the inhibitory effect on LPS-induced lation events (24). In conformity with these observations, our results IL-27 production following silencing of TRAF2, keeping in view show that macrophages employ the SHP-1/Src complex down- that LPS-induced IL-27 production is regulated by the canonical stream of TLR4 signaling to positively regulate IL-27 secretion pathway. through transcriptional regulation of its subunits. The mechanism of TRAF1 and 2 inhibitions may be an indirect Inflammasomes, multiprotein complexes of the innate immune effect of SMM on NF-kB signaling. Similar to cIAP1/2, TRAF1/2 system, play a key role in initiating inflammatory responses are also targets of SMM on NF-kB activity (64). Therefore, by during infection (69). Their nature and exact composition depend inhibiting LPS-induced classical NF-kB pathway, SMM may in- on the microbial activator (70). This study has provided evidence directly prevent TRAF1/2 induction. TRAF2 is a dual regulator of for the formation of a multiprotein complex encompassing SHP-1, NF-kB that promotes activation of the classical pathway, but in- Src, and the TLR4-associated proteins including cIAP1/2, TRAF2, hibits the noncanonical pathway (60) by inducing degradation of and MyD88 leading to IL-27 production in human macrophages. NIK (65). Therefore, SMM-induced TRAF2 downregulation may We and others have shown that MAPKs and PI3K regulate IL-27 also potentiate its effects on NF-kB pathways by promoting in- production to other stimuli (37, 71). Therefore, we investigated hibition of the classical pathway. whether the cIAP1/2–TRAF2–SHP-1–Src–MyD88 complex reg- The PTP SHP-1 plays a critical role in protection against au- ulates LPS-induced IL-27 production through the activation of toimmunity and inflammation and in the regulation of innate and MAPK/PI3K pathway. Our results show that IAP abolition with adaptive immune responses in murine models (26, 66). By SMM reduced LPS signaling through p38 and JNK MAPK and employing the me/me mice, we and others have shown SHP-1 as a PI3K pathways, suggesting that the signaling complex regulates key positive regulator of IL-6 and IL-10 in murine bone marrow– LPS-induced IL-27 production by inhibition of MAPKs and Akt/ derived macrophages (26) as well as in IL-21 production by CD4+ PI3K signaling. This study and our previous observations (37, 72) T cells resulting in a dampened Th17 response (67). In this study, show that PI3K pathway positively regulates IL-27 production in 1604 cIAP1/2–TRAF2–SHP-1–Src–MyD88 COMPLEX AND IL-27 REGULATION Downloaded from http://www.jimmunol.org/

FIGURE 10. Proposed model of regulation of LPS-induced IL-27 production in human primary macrophages. LPS binding to TLR4 triggers activation of MyD88 and formation of a signaling multiprotein complex comprising cIAP1/2–TRAF2–SHP-1–Src–MyD88, which activates downstream signaling pathways p38, JNK, and Akt. These kinases promote IkBa phosphorylation, thus targeting it for ubiquitination and activating classical NF-kB pathway, which leads to IL-27 protein synthesis. SMM inhibit this pathway by downregulating cIAP1/2 levels, which leads to decreased p38, JNK, and Akt signaling, inhibition of classical NF-kB pathway, and ultimately decreased IL-27 production. Alternatively, SMM stabilize NIK in the alternative NF-kB pathway, by guest on September 26, 2021 which promotes p100 accumulation and cleavage to p52, thus activating alternative NF-kB signaling. However, this effect does not seem to contribute to IL-27 regulation. human macrophages. However, PI3K pathway did not affect IL-27 monocytic cells (34, 35, 55). The effect on alternative NF-kB production in murine DCs (21).Thus, the role of PI3K pathway in pathway can be explained by cIAP ability to bind and degrade IL-27 production may vary from cell to cell type and the stimulus NIK (33). In the absence of cIAP1/2 as a result of SMM involved. treatment, NIK regulatory complex formation is im- In tumor cell lines SMM alone activate both classical and paired resulting in NIK accumulation and increased p100 to alternative NF-kB pathways by promoting TNF-a secretion p52 processing. and stabilizing NIK, respectively (38, 39). cIAP ablation also In summary, our results clearly show that SMM inhibit LPS- reduces activation of the classical NF-kB pathway in response induced IL-27 production in human macrophages without to members of the TNFR family, such as TNF-a (73, 74). impacting the cytokine production in the absence of TLR4 stim- However, the effect of SMM on NF-kBactivityinprimary ulation. Moreover, we have elucidated the signaling pathways that macrophages, either alone or in response to other immune regulate IL-27 production through the formation of a large sig- activators, remains unknown. We show that SMM alone did naling complex that brings together major signaling pathways, such not activate either classical or alternate NF-kB pathway in as SHP-1, Src, cIAPs, TRAF2, and MyD88. This signaling complex human macrophages. In contrast, SMM exhibited divergent impacts on p38 and JNK MAPK and PI3K and ultimately the effects on LPS-induced NF-kB signaling, causing inhibition of classical NF-kB pathway to regulate LPS-induced IL-27 produc- the classical NF-kB pathway and activation of the alternative tion in human macrophages (Fig. 10). These results assume noncanonical NF-kB pathway, manifested by reduced IkB significance when evaluating the clinical implications of SMM degradation and increased p100 processing, respectively. The administration, given the constant interest in SMM as cancer different results obtained in cell lines and primary cells on treatment. NF-kB activation may be due to different TNF-a secretion patterns in response to SMM. SMM-sensitive cell lines secrete Acknowledgments TNF-a in response to cIAP ablation and consequently become We thank Dr. M. Saxena for critically reading the manuscript. We also thank susceptible to TNF-a–induced apoptosis (38, 45). However, Dr. Bruno Fonseca for help in immunoprecipitation experiments. we did not detect enhanced TNF-a secretion following SMM treatment either alone or in response to LPS (Fig. 9B). This result is in keeping with the fact that SMM treatment causes Disclosures apoptosis of sensitive cancer cells (38, 39) but not of primary The authors have no financial conflicts of interest. The Journal of Immunology 1605

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