IL-1R−Associated -1 Mediates C δ-Induced IL-1β Production in Monocytes

This information is current as Rajiv Lochan Tiwari, Vishal Singh, Ankita Singh and Manoj of October 1, 2021. Kumar Barthwal J Immunol published online 29 July 2011 http://www.jimmunol.org/content/early/2011/07/29/jimmun ol.1002526 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 29, 2011, doi:10.4049/jimmunol.1002526 The Journal of Immunology

IL-1R–Associated Kinase-1 Mediates Protein Kinase Cd-Induced IL-1b Production in Monocytes

Rajiv Lochan Tiwari, Vishal Singh, Ankita Singh, and Manoj Kumar Barthwal

The role of IL-1R–associated kinase (IRAK)1 and its interaction with protein kinase C (PKC)d in monocytes to regulate IL-1b production has not been reported so far. The present study thus investigates such mechanisms in the THP1 cell line and human monocytes. PMA treatment to THP1 cells induced CD11b, TLR2, TLR4, CD36, IRAK1, IRAK3, and IRAK4 expression, IRAK1 kinase activity, PKCd and JNK phosphorylation, AP-1 and NF-kB activation, and secretory IL-1b production. Moreover, PMA- induced IL-1b production was significantly reduced in the presence of TLR2, TLR4, and CD11b Abs. Rottlerin, a PKCd-specific inhibitor, significantly reduced PMA-induced IL-1b production as well as CD11b, TLR2 expression, and IRAK1–JNK activation. In PKCd wild-type overexpressing THP1 cells, IRAK1 kinase activity and IL-1b production were significantly augmented, whereas recombinant inactive PKCd and PKCd small interfering RNA significantly inhibited basal and PMA-induced IRAK1

activation and IL-1b production. Endogenous PKCd–IRAK1 interaction was observed in quiescent cells, and this interaction was Downloaded from regulated by PMA. IRAK1/4 inhibitors, their small interfering RNAs, and JNK inhibitor also attenuated PMA-induced IL-1b production. NF-kB activation inhibitor and SN50 peptide inhibitor, however, failed to affect PMA-induced IL-1b production. A similar role of IRAK1 in IL-1b production and its regulation by PKCd was evident in the primary human monocytes, thus signifying the importance of our finding. To our knowledge, the results obtained demonstrate for the first time that IRAK1 and PKCd functionally interact to regulate IL-1b production in monocytic cells. A novel mechanism of IL-1b production that involves TLR2, CD11b, and the PKCd/IRAK1/JNK/AP-1 axis is thus being proposed. The Journal of Immunology, 2011, 187: 000–000. http://www.jimmunol.org/

nterleukin-1b is an important proinflammatory cytokine that aa region in their cytoplasmic tail called the Toll-IL-1R domain, has a role in varied types of diseases, including type 1 and which plays an important role in inducing the signals from this I type 2 diabetes, atherosclerosis, metabolic syndrome, and receptor (9). Consequently, signaling induced by these receptors is autoimmune diseases such as rheumatoid arthritis and inflam- very similar and culminates into activation of the NF-kB and JNK/ matory bowel disease (1–3). IL-1b is induced by TLRs or by AP-1 pathways (9–11). IL-1b induces the activation of NF-kBand cytokines such as TNF or IL-1b itself (2, 3). Before secretion, the JNK pathways by recruiting MyD88, IRAK1, TNFR-associated inactive form of the cytokine is processed by caspase-1, leading factor 6, and TGF-b–activated kinase 1 (1, 9). IL-1b–induced by guest on October 1, 2021 to its maturation and secretion (2, 3). Blood monocytes, tissue JNK and NF-kB activation diverges at IRAK (12). The IRAK macrophages, and dendritic cells are the major source of IL-1b; family consists of four members, namely IRAK1, IRAK2, IRAK3 however, NK cells and B lymphocytes also produce this cytokine (IRAKM), and IRAK4. IRAK1, IRAK2, and IRAK4 positively (1). Monocyte-derived resident macrophages often play a crucial regulate the immune response, and IRAKM usually antagonizes role in the generation of inflammatory responses in atherosclerosis their effect by disrupting the IRAK1/TNFR-associated factor 6 (4) and diabetes (1–3). Cytokines such as IL-1b secreted by these complex (7, 9). Out of all of these , IRAK1 and IRAK4 are cells often amplify the inflammatory response leading to lesion more worked-out proteins and are said to be true kinases, although progression and b cell death. IL-1b Ab is positioned for the their kinase activity is still under investigation (9). IRAK4 activates treatment of such disorders (5). IRAK1 by phosphorylating Thr387 and Ser376 present in the latter The IL-1R–associated kinase (IRAK) family of proteins repre- activation loop (13). Both IRAKM and IRAK2 are predicted to be sents important mediators of innate immunity and plays a crucial inactive kinases (9, 14), although recent work has shed more light role in the signaling cascade induced by the TLR/IL-1R family (6– on IRAK2 activity and its role in TLR signaling (14, 15). IRAK1- 9). Members of the TLR and IL-1R family possess a common 200- deficient macrophages and fibroblasts show impaired cytokine pro- duction in response to LPS and IL-1b (8, 16, 17). Division of Pharmacology, CSIR-Central Drug Research Institute, Council of Scien- Although previous studies demonstrate the role of IRAK1 in tific and Industrial Research, Lucknow 226 001, India TLR/IL-1R–induced signaling events (17), its role and regulation Received for publication July 28, 2010. Accepted for publication June 24, 2011. during IL-1b production are not known. It has been demonstrated This work was supported by the Department of Science and Technology, India (to that IRAK1 interacts and phosphorylates PKCi and regulates the M.K.B.) and by Fellowships from the Indian Council of Medical Research (to V.S.), NF-kB pathway (18). At the same time, PKCz also plays an im- the Council of Scientific and Industrial Research (to R.L.T.), and the University Grants Commission (to A.S.). This is CDRI communication number 8090. portant role in endotoxin-induced macrophage activation by acti- vating the TLR4/IRAK1 pathway (19). Furthermore, previous Address correspondence and reprint requests to Dr. Manoj Kumar Barthwal, Division of Pharmacology, CSIR-Central Drug Research Institute, 1 M.G. Marg, Lucknow 226 studies suggest a role of PKCd in IL-1b production from mono- 001, India. E-mail address: [email protected] cytes (20), but its interaction with IRAK1 is not known. Rottlerin, The online version of this article contains supplemental material. a PKCd-specific inhibitor, affects signaling events and cytokine Abbreviations used in this article: EGFP, enhanced GFP; INH, inhibitor; IRAK, IL- production in human monocytes stimulated by PMA and LPS 1R–associated kinase; MBP, myelin basic protein; PKC, protein kinase C; siRNA, (20). Therefore, the present study was undertaken to elucidate the small interfering RNA; WT, wild-type. role of the IRAK family of proteins in IL-1b production. At the Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 same time, we checked the hypothesis that PKCd could regulate

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002526 2 IRAK1 REGULATES IL-1b PRODUCTION Downloaded from

FIGURE 1. PMA induces time-dependent CD11b, TLR2, TLR4, and CD36 expression and IL-1b production. A, THP1 cells were stimulated with PMA http://www.jimmunol.org/ for different times and IL-1b production was measured in culture media by ELISA. B, CD11b, (C) TLR2, (D) TLR4, and (E) CD36 expression on THP1 cells was measured by flow cytometry after PMA stimulation for 24 and 72 h. Values represent means 6 SE. *p , 0.05, **p , 0.01, ***p , 0.001 versus control; ##p , 0.01, ###p , 0.001.

IRAK1 for IL-1b production. In this study we report the role of regulated by PKCd. With this finding IRAK1 can be positioned as IRAK family members in PMA-induced IL-1b production and an attractive target for regulating IL-1b production in various a novel IRAK1-mediated pathway of IL-1b production that is metabolic and autoimmune disorders. by guest on October 1, 2021

FIGURE 2. Time-dependent expression of IRAKs. A, THP1 cells stimulated with PMA for different time points were analyzed for IRAK1, IRAK2, IRAK3, and IRAK4 expressions by Western blotting. B, Densitometric analysis of the expressed IRAK isoforms in relative image quant units. C, IRAK1 kinase activity measured in an in vitro kinase assay at different times of PMA treatment. Cells were lysed, and immunoprecipitated IRAK1 was subjectedto kinase assay in the presence of [g-32P]ATP and MBP as substrate. Values represent means 6 SE. *p , 0.05, **p , 0.01, ***p , 0.001 versus control. The Journal of Immunology 3

Materials and Methods Human monocyte isolation, cell culture, and treatments Materials Human primary circulating monocytes were isolated as described earlier (21) with slight modification from healthy donors after their informed Pharmacological inhibitors, including IRAK1/4 inhibitor (INH), JNK INH 3 II, rottlerin, NF-kB activation INH, SN50, Go6976, Ro-31-8220, caspase- consent. Whole blood was centrifuged at 250 g for 20 min and the upper 1 INH VI (Z-YVAD-FMK), and Pam3CSK4 were purchased from Cal- layer rich in platelets (platelet-rich plasma) was removed. Remaining 3 biochem (San Diego, CA). PMA, LPS, SB 202190, myelin basic protein blood was centrifuged at 650 g for 20 min and buffy coats were col- (MBP), protease inhibitor mixture, Abs against human IRAK1, IRAK2, lected, which were mixed with saline and subjected to dextran sedimen- and IRAK3, and b-actin were from Sigma-Aldrich (St. Louis, MO). tation. The upper layer rich in leukocytes was collected and centrifuged at IRAK1, IRAK4, p-IRAK, p-PKCd, and PKCd Abs were procured from 500 3 g for 5 min at room temperature. Pellets were resuspended in HBSS Cell Signaling Technology (Danvers, MA). Human anti–CD36-FITC, containing glucose. Density gradient centrifugation utilizing Percoll 1080 anti–p-JNK, and anti-total JNK were from Millipore (Billerica, MA). and 1065 was done at 700 3 g for 15 min, and the interface layer was Tanshinone IIa, human anti–TLR2-rPE, human anti–TLR4-FITC, human collected and washed with glucose HBSS. Pellet was resuspended in RPMI anti–IL-1b and anti–CD11b-rPE, and control small interfering RNA 1640 and loaded on a hyperosmotic gradient, and the interface layer of (siRNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, monocytes was adhered in RPMI 1640 containing 10% FBS for 1 h and CA). IRAK siRNAs were purchased from Santa Cruz Biotechnology and subsequently used for experiments (21). Viability of cells was found to be Dharmacon (Chicago, IL). ECL reagent was from GE Healthcare (Pis- .95% as assessed by trypan blue staining, and purity of cells was found to cataway, NJ). Tissue culture reagents were procured from Invitrogen be .95% as assessed by CD14+ cells by flow cytometry. In addition to this (Carlsbad, CA). All other fine chemicals used in the study were procured human monocytic cell line, THP1 cells were cultured in RPMI 1640 from Sigma-Aldrich. containing 10% heat-inactivated FBS, 100 IU/ml penicillin, and 100 mg/ml Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. IRAKs mediate IL-1b production in THP1 cells. PMA-induced IL-1b production in THP1 cells was measured after (A) pretreatment with IRAK1/4 INH (0.3 mM) and transfection of (B) IRAK1 siRNA, (C) IRAK2 siRNA, (D) IRAK3 siRNA, and (E) IRAK4 siRNA. Culture supernatant was used to measure IL-1b by ELISA whereas cells were lysed and analyzed by Western blotting for the expression of various IRAK isoforms. Values represent means 6 SE. **p , 0.01, ***p , 0.001 versus control; ###p , 0.001. 4 IRAK1 REGULATES IL-1b PRODUCTION streptomycin. Monocytic cells were treated for 5, 15, 30, 60 min and 1, 6, (BD OptEIA set human IL-1b; BD Biosciences, San Diego, CA) as per the 12, 12, 48, and 72 h with PMA (200 nM) (22, 23), or for 24 h with LPS or manufacturer’s protocol. In brief, supernatants were collected from PMA- Pam3CSK4 (100 ng/ml). As required, cells were also pretreated for 1 h treated THP1 cells and incubated for 2 h at room temperature in overnight with different pathway inhibitors and Abs at reported concentrations be- capture Ab-coated ELISA plates. After incubation, wells were washed fore PMA/LPS or Pam3CSK4 treatment. Inhibitors used in the present with PBS containing 0.05% Tween 20 and incubated with detection Ab study were IRAK1/4 INH (0.3 mM), JNK INH 2 (10 mM), general PKC followed by washing and reagent incubation. The color was de- INH (Ro-31-8220; 1 mM), classical PKC INH (Go6976; 20 nM), PKCd veloped by adding a TMB substrate reagent set (BD Biosciences) and INH (rottlerin; 2 mM), p38MAPK INH (SB 202190; 0.2 mM), NF-kB subsequently read at 450 and 570 nm on an ELISA plate reader (Bio-Tek activation INH (small molecule; 20 nM), NF-kB peptide INH (SN50; Instruments, Winooski, VT). Standard IL-1b provided in the was used 1 mM), caspase-1 INH VI (1 mM), and AP-1 INH (tanshinone IIa; 1 mM). for drawing the standard and calculation of absolute IL-1b levels. Treatments with CD36, TLR2, TLR4, CD11b, and isotype control Abs were given at 5 mg/ml. Western and phospho-blotting Assay for IL-1b production Cells were harvested after desired treatments and lysed in lysis buffer containing 0.1 M NaCl, 0.01 M Tris-HCl (pH 7.4), 0.001 M EDTA (pH 7.4), Production of secretory IL-1b after treatment with PMA, different pathway aprotinin (1 mg/ml), PMSF (100 mg/ml), pepstatin (20 mg/ml), sodium inhibitors, and Abs was measured in the media by conventional ELISA orthovanadate (2 mM), sodium fluoride (2 mM), and 1% Triton X-100. The Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. IRAK-dependent IL-1b production in primary human monocytes. PMA-induced IL-1b production from primary human monocytes was measured after (A) pretreatment with IRAK1/4 INH (0.3 mM) and transfection of (B) IRAK1 siRNA, (C) IRAK2 siRNA, (D) IRAK3 siRNA, and (E) IRAK4 siRNA. Culture supernatant was used to measure IL-1b by ELISA whereas cells were used to analyze the expression of IRAKs by immunoblotting. Values represent means 6 SE. **p , 0.01 versus control; #p , 0.05. The Journal of Immunology 5 cell extracts were clarified at 15,000 3 g for 5 min, and protein contents AP-1 and NF-kB activity assays were measured by using BCA reagent. Equal amounts of lysate were boiled in Laemelli buffer and separated on denaturing 7–10% SDS-PAGE and AP-1 and NF-kB activities were measured at different time points of PMA transferred onto polyvinylidene difluoride membranes. After blocking treatment by using commercially available ELISA kit (TransAM AP-1-c- (5% BSA in TBST), the membrane was incubated with primary Abs against Jun and TransAM NF-kB-p65; Active Motif, Carlsbad, CA). Nuclear ex- various candidate proteins such as IRAKs (1:1000), p-IRAK (1:1000), tracts were prepared as per kit instructions. Briefly, monocytic cells after p-JNK (1:1000), JNK (1:1000), p-PKCd (1:500), PKCd (1:1000), IL-1b treatment were collected and washed with ice-cold phosphatase inhibitor (1:1000), and b-actin (1:2000) as per the manufacturer’s protocol. This was buffer (125 mM NaF, 250 mM b-glycerophosphate, 250 mM para-nitro- followed by incubation with specific HRP-conjugated secondary Ab. The phenyl phosphate, 25 mM NaVO3) and resuspended in 1 ml ice-cold hy- specific bands were detected by ECL as described earlier (24, 25). potonic buffer (20 mM HEPES [pH 7.5], 5 mM NaF,1 0mMNa2MoO4, 0.1 mM EDTA). The cells were allowed to swell for 15 min on ice. Fifty Immunoprecipitation and in vitro kinase assay microliters of 10% Nonidet P-40 was added and the tube was shaken for 10 s. Cell homogenate was centrifuged for 30 s at 4˚C and supernatant (cy- IRAK1, IRAK2, and IRAK4 kinase assays were performed as described toplasmic fraction) was removed. Nuclear pellet was suspended in 50 ml earlier (18, 24). Cells from different experimental groups were lysed in complete lysis buffer for 30 min at a rocking platform. The lysate was 0.1% Nonidet P-40 lysis buffer (50 mM Tris-Cl [pH 8.0], 137 mM sodium centrifuged at 15,000 3 g at 4˚C for 10 min and an equal amount of chloride, 2 mM EDTA, 5% glycerol, 0.1% Nonidet P-40) supplemented nuclear extract was used for AP-1 (c-Jun) and NF-kB (p65) assays after with 1:100 protease inhibitor mixture. The lysates were centrifuged at protein quantification. Both AP-1and NF-kB were measured by loading 15,000 rpm, supernatants were collected, and protein concentration was 10 mg nuclear extract onto a well of 96-well microtiter plates coated measured. Preclearing of cell lysate was performed by incubating 400 mg with oligonucleotides 59-TGAGTCA-39 and 59-GGGACTTTCC-39, respec- cell extracts from different experimental groups with 20 ml protein A- tively, for 1 h. After washing three times, mAbs against c-Jun and p65 Sepharose beads (50% slurry) for 45 min at 4˚C. After centrifugation at 3 were added to appropriate wells and incubated for a further 1 h at room 14,000 g for 10 min, the supernatant was mixed with 2.0 mg/ml rabbit temperature. Anti–IgG-HRP conjugate in a volume of 100 ml was then anti-IRAK1, -IRAK2, and -IRAK4 Abs and incubated at 4˚C overnight. added and further incubated for 1 h at 25˚C. Absorbance at 450 nm was Downloaded from Subsequently, 20 ml protein A-Sepharose beads (50% slurry) was mixed measured after the addition of tetramethylbenzene solution. Absolute and further rotated for 2 h at 4˚C. The protein A-Sepharose beads were levels of the transcription factor were quantified by setting up standard spun down and washed four times with lysis buffer and twice with 0.1 M curves by the help of reagents provided in the kit. LiCl. The immunoprecipitates were processed for immuno-blotting as desired. For kinase assays, the immunocomplexes were washed with ki- siRNA and plasmid transfections nase assay buffer (20 mM MOPS [pH 7.2], 50 mM MgCl2, 2 mM EGTA, 1 mM DTT) (24). Reaction was carried out in the presence of 5 mg MBP Transfections were performed by using an Amaxa Nucleofector machine 32 substrate, 0.5 mM ATP, and 10 mCi [g- P]]ATP for 30 min at 30˚C (Amaxa, Cologne, Germany) as described earlier (25) and in the optimized http://www.jimmunol.org/ (18, 24). Reactions were stopped by the addition of 15 ml63 SDS-PAGE protocol for THP1 and primary monocytes provided by the manufacturer. sample buffers and subsequently boiled. Supernatants were subjected Briefly, 1 3 106 THP1 cells in 100 ml transfection reagent provided in to SDS-PAGE and transferred to polyvinylidene difluoride membranes. kit (Cell Line Nucleofector Kit V) were transfected with 3.0 mg control Phosphorylation of the substrate was measured by autoradiography. or IRAK1, IRAK2, IRAK3, and IRAK4, PKCd siRNA by using the by guest on October 1, 2021

FIGURE 5. PMA-induced IL-1b production is JNK dependent but independent of NF-kB. THP1 cells were stimulated with PMA for different times and processed for the measurement of various parameters. A, NF-kB activity in nuclear extracts was measured by TransAM NF-kB-p65 kits. B, JNK activation was measured by probing the blots with activated p-JNK Ab that recognizes both pJNK1 and pJNK2. Blots were also probed with total JNK and b-actin Ab. C, AP-1 activity was measured in nuclear extracts by using a TransAM AP-1-c-Jun kit. D, IL-1b production was measured in PMA-stimulated THP1 cells that were pretreated with JNK INH II, SN50, NF-kB activation INH, LY294002, and SB202190. Values represent means 6 SE. *p , 0.05, **p , 0.01, ***p , 0.001 versus control; #p , 0.05, @@@p , 0.001 versus PMA 30 min; $$$p , 0.001 JNK1 versus JNK2 at indicated time points. 6 IRAK1 REGULATES IL-1b PRODUCTION

FIGURE 6. PKCd mediates PMA-induced IL- 1b production. A, Phosphorylation of PKCd in THP1 cells was measured at various time points by Western blotting. Cell extracts were resolved on SDS-PAGE and after transfer to polyviny- lidene difluoride membrane were probed with a phosphorylated Ab that recognizes activated PKCd. At the same time, expression of total PKCd and b-actin was monitored by probing the blots with specific Abs. B, IL-1b production at 24 h in PMA-stimulated THP1 cells was mea- sured after pretreatment with rottlerin. Values re- present means 6 SE. *p , 0.05, ***p , 0.001 versus control; ###p , 0.001.

Nucleofector machine program V001. After transfection, cells were re- Flow cytometry moved in 0.5 ml RPMI 1640 and plated in 1 ml prewarmed medium in 6- well plates. After 1 h transfection, PMA treatment was given and IL-1b was Flow cytometric measurements were performed using a three-color 6 3 6 Downloaded from subsequently measured at 24 h. Monocytes (4 3 10 ) were also transfected FACSCalibur (BD Biosciences, San Jose, CA). THP1 cells (1 10 ) after with 3.0 mg control or IRAK1, IRAK2, IRAK3, and IRAK4 siRNA by using different treatments were harvested and washed twice with PBS. For detec- 2+ 2+ the Human Monocyte Nucleofector Kit and program Y001. Similar methods tion of surface markers, cells were resuspended in Ca -Mg HBSS and in- were used to transfect the p-enhanced GFP (EGFP)-N1 vector, p-EGFP-N1- cubated with conjugated primary Abs (TLR2-rPE, TLR4-FITC, CD11b-rPE, PKCd-wild type (WT), and p-EGFP-N1-PKCd-K376R constructs. Expres- and CD36-FITC) for 30 min in the dark at 37˚C as described earlier (26, 27). sion of recombinant GFP provided in the kit and FITC-labeled control After labeling, cells were washed with PBS and resuspended in 1 ml Ca2+- siRNA were used as markers for monitoring the transfection efficiency. Mg2+ HBSS, and 10,000 events were analyzed by FACS. CellQuest was used

Gene silencing was measured after 24 h by Western blotting. for quantifying the expression of various receptors. http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 7. PKCd operates upstream of IRAK1 and JNK during IL-1b production. Rottlerin-pretreated THP1 cells were harvested after 15 min PMA treatment and processed for IRAK1 activation (A–C). A, Phoshorylation of IRAK1 was measured by immunobloting with p-IRAK1 Ab. Total IRAK1 expression was ascertained by probing with total IRAK1 Ab. B, IRAK1 kinase activity was measured by in vitro kinase assay using [g-32P]ATP and MBP as substrate. C, Modulation of interaction between IRAK1 and PKCd assessed after immunoprecipitation with anti-IRAK1 and corresponding IgG isotype control and immunoblotting with anti-PKCd. A portion of the cell lysate was also processed for the evaluating total PKCd expression. D, Phoshorylation of JNK was measured in rottlerin- or IRAK1/4 INH-pretreated THP1 cells by phospho-blotting after 15 min PMA treatment. Membranes were also probed with total JNK and b-actin Abs. E, THP1 cells were pretreated with rottlerin, JNK INH II, IRAK1/4 INH, and their combinations. Subsequently, secreted IL-1b was measured after 24 h PMA stimulation. Values represent means 6 SE. ***p , 0.001 versus control; #p , 0.05, ##p , 0.01, ###p , 0.001, $$$p , 0.001 versus PMA alone. The Journal of Immunology 7

Caspase-1 fluorometric assay method. Actin was used as internal standard to calculate the relative ex- pression (31). Caspase-1 activity was assayed by using a caspase-1 fluorometric assay kit (R&D Systems, Minneapolis, MN). After various treatments, cells were Statistical analysis collected by centrifugation at 250 3 g for 10 min. Kit buffer was used for cell lysis. Supernatant obtained after centrifugation at 10,000 3 g was Results are expressed as the means 6 SE. Comparisons were done by used for caspase-1 assay. Total protein (200 mg) was mixed with an equal a Bonferroni multiple comparison test or by one-way ANOVA for multiple volume of 23 reaction buffer in a microplate. Reactions were initiated comparisons of three or more groups. The differences were considered by the addition 5 ml caspase-1 fluorogenic substrate (WEHD-AFC). Re- significant with a p value ,0.05. Blots represent one of three or more action was carried out at 37˚C for 2 h. Plates were read at excitation of similar experiments. 400 nm and emission of 505 nm in an LS 55 fluorescence plate reader (PerkinElmer, Wellesley, MA). The results were expressed as fold increase in caspase-1 activity of induced cells over that of noninduced cells (28). Results Monocyte activation and secretory IL-1b production Expression of IL-1b by real-time PCR For studying monocyte activation and innate immune response Total RNA from THP1 cells was extracted by using TRI Reagent. For quantitative RT-PCR analysis of IL-1b,cDNAwassynthesizedfrom1mg during PKCd activation, we used PMA to stimulate our cells. Se- RNA by using commercially available cDNA synthesis kit (RevertAid first cretory IL-1b production from THP1 cells was measured in the stand DNA synthesis kit; Fermentas Life Sciences). Real-time PCR was media after different times of PMA treatment (Fig. 1A). A signifi- done in a 25 ml reaction by using Maxima SYBR Green/ROX qPCR Master cant increase in IL-1b production was observed from 1 h PMA 3 Mix (2 ) (Fermentas Life Sciences), IL-1b- (forward primer, 59-CTCT- treatment (Fig. 1A). At 6 h the increase was almost 6-fold more than CTCACCTCTCCTACTCAC-39, reverse primer, 59-ACACTGCTACTTCT- TGCCCC-39)andb-actin- (forward primer, 59-AACTGGAACGGTGAA- at 1 h. This increase was sustained until 24 h. Subsequently, a ten- GGTG-39, reverse primer, 59-CTGTGTGGACTTGGGAGAGG-39)specific dency of decrease was observed at 48 and 72 h. Although the de- Downloaded from primers, and a LightCycler 480 real-time PCR system (Roche Applied crease in IL-1b production at 72 h was significantly less than those Science, Mannheim, Germany). A three-step cycling protocol (initial de- at 12 and 24 h, it was still significantly more than the control levels naturation at 95˚C for 10 min, 35 cycles of 15 s denaturation at 95˚C, 30 s A annealing at 60˚C, and 30 s extension at 72˚C) was used to amplify the (Fig. 1 ). As markers for monocyte activation, differentiation, and (29, 30). Relative fold difference between an experimental and calibrator induction of an innate immune response, expression of CD11b, sample were calculated by using the comparative cycle threshold (22ΔΔCt) TLR2, TLR4, and CD36 on THP1 cells was measured by flow http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 8. Role of the PKCd/IRAK1/JNK axis in PMA-induced IL-1b transcription and caspase-1 activation. Rottlerin, JNK INH II-, IRAK1/4 INH-, or caspase-1 INH-pretreated THP1 cells were stimulated with PMA for 24 h. Subsequently, (A) cellular IL-1b mRNA by quantitative RT-PCR, (B) Pro and mature IL-1b protein expression by Western blotting, (C) caspase-1 activity, and (D) secreted IL-1b levels in the medium by ELISA were measured. Values represent means 6 SE. *p , 0.05, **p , 0.01, ***p , 0.001 versus control; ###p , 0.001, $$$p , 0.001 versus PMA alone. 8 IRAK1 REGULATES IL-1b PRODUCTION cytometry at 24 and 72 h PMA treatment. A significant increase in production. Elevated IRAK1 kinase activity (∼2-fold) was observed expression of CD11b (Fig. 1B), TLR2 (Fig. 1C), TLR4 (Fig. 1D), after 5 and 15 min PMA treatment of THP1 cells (Fig. 2C). and CD36 (Fig. 1E) was observed at 24 and 72 h, indicating the For ascertaining the role of IRAK1 in IL-1b production, we activation of monocytes and innate immune signaling cascade. carried out experiments in the presence of IRAK1/4 INH. A sig- Expression levels of CD11b (Fig. 1B) and TLR4 (Fig. 1D)at72h nificant inhibition (p , 0.001) in PMA-induced IL-1b production were significantly more than those at 24 h. Although a tendency of was observed in IRAK1/4 INH-pretreated THP1 cells (Fig. 3A). increase in the expression of CD36 (Fig. 1E) and TLR2 (Fig. 1C) Isoform-specific siRNA and scrambled control siRNA were trans- was observed from 24 to 72 h, it was not statistically significant. fected by nucleofection for elucidating the role of individual IRAK isoforms in IL-1b production. Reduction in expression of IRAK b Activation and involvement of the IRAK pathway during IL-1 isoforms was observed on specific siRNA treatment (Fig. 3B–E). production IRAK1 siRNA-mediated gene silencing significantly inhibited Because the IRAK family of proteins mediates innate immune PMA-induced IL-1b production in THP1 cells (Fig. 3B). IRAK2 response (9), we evaluated the role of these proteins in IL-1b siRNA also significantly inhibited PMA-induced IL-1b pro- production under present experimental conditions. As IRAK1 duction (Fig. 3C). There was no significant effect of IRAKM activation is often accompanied with its degradation (32), we siRNA on PMA-induced IL-1b production (Fig. 3D). IRAK4 monitored the expression of all IRAK isoforms by Western blot- siRNA showed similar kinds of inhibition (Fig. 3 E). To ascertain ting at different times of PMA treatment. Significant increase in whether the IRAK family of proteins had a similar role in primary IRAK1 expression was observed after 30–60 min PMA treatment. cells, we conducted experiments in human monocytes. For this, Maximum induction was seen at 6 h (Fig. 2A,2B). Subsequently, IRAK1/4 INH and gene-specific siRNA was used to inhibit IRAK- a decrease in IRAK1 expression was observed (Fig. 2A,2B). mediated signaling events. Pretreatment with IRAK1/4 INH sig- Downloaded from Induction of IRAK4 was also observed on PMA treatment that nificantly reduced PMA-induced IL-1b production (Fig. 4A). peaked at 30 min to 1 h and thereafter stabilized (Fig. 2A,2B). siRNA-mediated specific gene silencing was conducted in primary Expression of IRAK2 was unaltered on PMA treatment. A late monocytes (Fig. 4B–E). Significant reduction in IRAK1, IRAK2, induction in IRAKM was also observed from 6 to 12 h PMA IRAK3, and IRAK4 expression was observed on treating with treatment (Fig. 2A,2B). Because it has been reported that IRAK1 their specific siRNAs (Fig. 4B–E). Similar to THP1 cells, re- is downstream of IRAK4 (9), we performed kinase assays for the duction in IL-1b production was observed on treating the cells http://www.jimmunol.org/ former enzyme so as to ascertain the activation of this pathway and with IRAK1, IRAK2, or IRAK4 siRNAs (Fig. 4B, C, E). No re- whether IRAK1 kinase activity is important for inducing IL-1b duction was seen with IRAKM siRNA (Fig. 4D). by guest on October 1, 2021

FIGURE 9. Role of AP-1 in PMA-induced IL-1b transcription. Tanshinone IIa-pretreated THP1 cells were stimulated with PMA for 24 h. Subsequently, (A) cellular IL-1b mRNA by quantitative RT-PCR, (B) Pro and mature IL-1b protein by Western blotting, and (C) secreted IL-1b in the medium by ELISA were measured. Values represent means 6 SE. *p , 0.05, **p , 0.01, ***p , 0.001 versus control; ###p , 0.001, $$$p , 0.001 versus PMA alone. The Journal of Immunology 9

PMA induces IL-1b production in a JNK-dependent but levels at 24 and 48 h PMA treatment were significantly more NF-kB–independent manner than the control levels (Fig. 5B,5C). To elucidate the role of the BecauseIRAK1 signaling bifurcates downstream into an NF-kB JNK and NF-kB pathways in IL-1b production, the levels of se- or JNK pathway (12), we monitored their activation by ELISA creted cytokine were measured in the presence of JNK INH 2, NF- and phospho-blotting, respectively. A time-dependent activation kB activation INH, and inhibitor peptide SN50 (Fig. 5D). PMA- of NF-kB and JNK pathways was observed on treating the THP1 induced IL-1b production was significantly inhibited in the pres- cells with PMA (Fig. 5A,5B). An ∼7-fold induction in NF-kB ence of JNK INH 2 (Fig. 5D). However, neither cell-permeable k activity was seen at 12–24 h PMA treatment (Fig. 5A). Activation peptide inhibitor (SN50) that prevents translocation of NF- B into the nucleus nor the small molecule inhibitor (NF-kB activation of the JNK pathway was also observed (Fig. 5B). Both JNK1 and INH) inhibited PMA-induced IL-1b production. Therefore, al- JNK2 were significantly activated at all times of analysis (Fig. though we did observe NF-kB activation, it was not involved in 5B). Although the expression of JNK1 and JNK2 was similar, IL-1b production (Fig. 5D). Because PI3K and p38MAPK are JNK1 was significantly more phosphorylated than JNK2 at most known to play a role in IL-1b production (36–40) and IRAK can of the times analyzed (Fig. 5B). also activate p38MAPK (9), we evaluated PMA-induced IL-1b Initial hyperactivation at 15–30 min and late at 12–24 h PMA production in the presence of SB 202190 (p38MAPK INH) and treatment was observed (Fig. 5B). Although a significant decrease LY294002 (PI3K INH) (Fig. 5D). These pharmacological inhib- in JNK1 and JNK2 phosphorylation was observed at 1–6 h when itors failed to prevent PMA-induced IL-1b production (Fig. 5D). compared with 30 min, it was still significantly more than the Because PMA specifically activates PKC, we can conclude that control levels (Fig. 5B). Because AP-1 is downstream of JNK (11) PKC-mediated IL-1b production is independent of PI3K and and PMA-induced IL-1b production in THP1 cells is mediated Downloaded from p38MAPK, but this will need further confirmation. by AP-1 (33–35), it was logical to monitor its activation under d b present experimental conditions. Significant increases in nuclear The PKC /IRAK1/JNK module regulates IL-1 production AP-1 levels were observed at 30 min PMA treatment (Fig. 5C). Before analyzing the detailed role of PKCd in PMA-induced Interestingly, similar to JNK1 and JNK2, a significant decrease in IL-1b production, we confirmed its activation status by phospho- activated AP-1 levels was observed at 1–6 h, but this was still blotting. A time-dependent increase in PKCd phosphorylation was

significantly more than the control (Fig. 5C). Similar to JNK, AP-1 observed on PMA treatment (Fig. 6A). A significant increase could http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 10. IRAK1 mediates PKCd-induced IL-1b production in primary monocytes. A, Secreted IL-1b was measured in rottlerin-pretreated primary human monocytes after stimulation with PMA for 24 h. B–D, Rottlerin pretreated THP1 cells were harvested after 15 min PMA treatment and processed for measuring (B) p-IRAK by immunoblotting, (C) IRAK1 kinase activity in an in vitro kinase assay by utilizing [g-32P]ATP and MBP as substrate, and (D) modulation of IRAK1 and PKCd interaction by immunoprecipitation with anti-IRAK1 and corresponding IgG isotype control and immunoblotting with anti-PKCd. A portion of the cell lysate was also processed for evaluating total PKCd expression. Values represent means 6 SE. **p , 0.01,***p , 0.001 versus control; ##p , 0.01, ###p , 0.001. 10 IRAK1 REGULATES IL-1b PRODUCTION be seen from an early time point of 15 min and maximum phos- PMA (Fig. 7C). Rottlerin could inhibit this regulation (Fig. 7C). phorylation was observed at 1 h (∼8-fold) (Fig. 6A). Activation was To test whether this IRAK1 and PKCd regulation feeds into the observed at all the points of analysis (Fig. 6A). A significant inhibi- JNK pathway, we also assessed JNK activation (Fig. 7D). Signi- tion in PMA-induced IL-1b production was observed with PKCd ficant inhibition in phosphorylation of both JNK1 and JNK2 was specific inhibitor rottlerin (Fig. 6B), indicating the role of PKCd. observed with both rottlerin and IRAK1/4 INH, indicating that PMA-stimulated THP1 cells pretreated with a general PKC INH both isoforms may be involved in PKCd-IRAK1–mediated IL-1b Ro-31-8220 also showed decreased levels of secreted IL-1b pro- production. More importantly, this also established the existence duction (Supplemental Fig. 1); however, the classical PKC INH of the PKCd/IRAK1/JNK axis in PMA-induced IL-1b production Go6976 had no significant effect (Supplemental Fig. 1). (Fig. 7D). To ascertain whether components of the PKCd/IRAK1/ To determine whether inhibition of PKCd affects IRAK1, we JNK module also feed into other pathways for regulating IL-1b checked IRAK1 activation by performing phospho-blotting for ac- production, we measured the levels of this cytokine in the pres- tive IRAK1 and IRAK1 kinase activity assay in THP1 cells pre- ence of various combinations of rottlerin, IRAK1/4 INH, and JNK treated with rottlerin (Fig. 7A,7B). Rottlerin also inhibited PMA- INH (Fig. 7E). Synergistic inhibition of IL-1b production was induced IRAK1 phosphorylation (Fig. 7A) and kinase activity (Fig. observed on pretreatment with rottlerin-IRAK1/4 INH or rottlerin- 7B), indicating a positive role of PKCd in modulating IRAK1. JNK INH when compared with rottlerin or IRAK1/4 INH alone Although PMA also induced activation of IRAK2 (Supplemental (Fig. 7E). IRAK1/4 and JNK INH combination could inhibit IL- Fig. 2A) and IRAK4 (Supplemental Fig. 2B), their kinase activities 1b production significantly more than IRAK1/4 alone, indicating were unaffected by rottlerin (Supplemental Fig. 2). that JNK may also feed into other signaling pathways to induce At the same time, we did not observe any change in PKCd IL-1b production (Fig. 7E). Maximal inhibition was achieved phosphorylation in the presence of IRAK1/4 INH, confirming that when all three inhibitors were used together (Fig. 7E). Because Downloaded from PKCd is upstream of IRAK1 (data not shown). More importantly, levels of secreted IL-1b can be modulated by transcription, we observed a direct endogenous interaction between IRAK1 and translation, and processing, we performed real-time PCR (Fig. 8A) PKCd in quiescent monocytic THP1 cells that was modulated by and Western blotting (Fig. 8B) in THP1 cell lysates for assessing http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 11. PKCd siRNA and kinase-inactive PKCd suppresses IRAK1 activation. A, IRAK1 activity was measured in PKCd-siRNA–treated THP1 cells after PMA treatment for 15 min. A portion of cell lysate was also processed for analyzing the expression of PKCd and b-actin. B, THP1 cells were transfected with control vector p-EGFP-N1, active PKCd (p-EGFP-N1-PKCd-WT), and kinase-inactive PKCd (p-EGFP-N1-PKCd-K376R) and stimulated with PMA for 15 min for the measurement of IRAK1 kinase activity in an in vitro kinase assay by utilizing [g-32P]ATP and MBP as substrate. A portion of the cell lysate was also processed for analyzing the expression of recombinant PKCd by probing with GFP Abs. C, IL-1b production was also measured in the above samples by regular ELISA. Values represent means 6 SE. *p , 0.05, **p , 0.01, ***p , 0.001 versus control; ###p , 0.001. The Journal of Immunology 11

IL-1b mRNA and protein levels, respectively. For assessing the (Fig. 10A). Similar to THP1 cells, increases in IRAK1 phos- contribution of processing pathways in IL-1b production, a cas- phorylation (Fig. 10B) and activity (Fig. 10C) induced by PMA pase-1 assay was performed in lysates of THP1 cells (Fig. 8C). A were inhibited by rottlerin (Fig. 10B,10C). Most importantly, significant increase in IL-1b mRNA (Fig. 8A) and protein (Fig. endogenous interaction between IRAK1 and PKCd and modu- 8B) levels was observed after PMA treatment. Rottlerin, IRAK1/4 lation by PMA was also observed in primary monocytes (Fig. INH, and JNK INH significantly attenuated PMA-induced IL-1b 10D). mRNA (Fig. 8A) and protein levels (Fig. 8B), indicating that the inhibition in secreted IL-1b levels is due to attenuated IL-1b Recombinant PKCd regulates IRAK1 for IL-1b production transcription. However, at the same time a significant increase in To confirm the effect of PKCd, its kinase activity, and rottlerin caspase-1 activity was observed after PMA treatment (Fig. 8C). on endogenous IRAK1 activation, we assessed the effect of PKCd To assess the contribution of the PKCd/IRAK1/JNK axis in PMA- siRNA and recombinant kinase active and inactive PKCd on induced caspase-1 activation, we performed caspase-1 assays in IRAK1 activation and IL-1b production (Fig. 11). PKCd siRNA- the presence of rottlerin, IRAK1/4 INH, and JNK INH (Fig. 8C). treated cells displayed a significant inhibition in PMA-induced IRAK1/4 INH and JNK INH had no significant effect on caspase-1 IRAK1 activation (Fig. 11A), indicating a positive role of PKCd activation (Fig. 8c). However, rottlerin could significantly atten- in IRAK1 activation. uate caspase-1 activity (Fig. 8C). More importantly, treatment Overexpression of p-EGFP-N1-PKCd-WT in monocytic THP1 with caspase-1 INH significantly attenuated the levels of secreted cells induced a significant increase in IRAK1 kinase activity (Fig. IL-1b, indicating a caspase-1–dependent increase in IL-1b pro- B lane 6 b C cessing after PMA treatment (Fig. 8D). 11 , ) and IL-1 production (Fig. 11 ) when compared Because AP-1 mediates JNK-induced effects, its role in IL-1b with p-EGFP-N1 vector alone (Fig. 11B,11C). At the same time, Downloaded from production was also studied. Cellular IL-1b mRNA (Fig. 9A), overexpression of recombinant inactive p-EGFP-N1-PKCd-K376R protein (Fig. 9B), and secreted IL-1b (Fig. 9C) levels were eval- construct significantly reduced the basal IRAK1 kinase activity uated in presence of AP-1 INH transhinone IIa. PMA-induced (Fig. 11B, lane 4) and IL-1b production (Fig. 11C). IRAK1 kinase significant induction in cellular IL-1b mRNA, protein, and se- activity of PMA-stimulated THP1 cells overexpressing PKCd- creted IL-1b levels was significantly reduced in the presence of K376R was significantly less than PKCd-WT overexpressing this inhibitor (Fig. 9). cells, indicating that kinase activity of PKCd is required for in- http://www.jimmunol.org/ For ascertaining whether similar PKCd and IRAK1 regula- ducing IRAK1 activation (Fig. 11B, lane 6 versus lane 7) and IL- tion exists in primary cells, we performed experiments in human 1b production (Fig. 11C). These results confirmed that kinase monocytes. In primary monocytes PMA-induced increase in activity of PKCd is important for IRAK1-induced IL-1b pro- IL-1b production was inhibited on prior treatment with rottlerin duction. by guest on October 1, 2021

FIGURE 12. CD11b- and TLR2-mediated IL-1b production is dependent on PKCd. A, THP1 cells were preincubated with control IgG, anti-TLR2, anti- TLR4, anti-CD11b, and anti-CD36 Abs for 1 h and subsequently stimulated with PMA for 24 h. Secreted IL-1b was measured in the supernatant. B, CD11b, (C) TLR2, (D) TLR4, and (E) CD36 expression in rottlerin-pretreated and 24 h PMA-stimulated THP1 cells were analyzed by flow cytometry. Values represent means 6 SE. *p , 0.05, ***p , 0.001 versus control; ##p , 0.01, ###p , 0.001. 12 IRAK1 REGULATES IL-1b PRODUCTION

TLR2 and CD11b regulate secretory IL-1b production in upregulation of CD11b, CD36, TLR2, and TLR4 receptors. Up- a PKCd-dependent manner regulation of CD11b is often associated with differentiation of To ascertain the role of PKCd in regulating cell surface receptors human monocytes to macrophages (41, 42). This receptor is up- that are usually associated with monocyte activation and genera- regulated in the diabetic pancreas (43) and plays an important role tion of innate immune response, we studied the role of TLR2, in adhesion to the (44). Upregulation of this receptor TLR4, CD11b, and CD36 in IL-1b production. Pretreatment of is also seen during macrophage foam cell formation (45). Among THP1 cells with anti-CD11b, anti-TLR2, and anti-TLR4 Abs the TLR receptors, TLR2 and TLR4 are upregulated in lesion significantly reduced PMA-induced IL-1b production (Fig. 12A). macrophages (46). At the same time, enhanced expression of Maximum inhibition was seen with anti-CD11b and anti-TLR2 macrophage TLR is also observed in the diabetic pancreas (47). (∼40%) (Fig. 12A). Blocking the CD36 receptor had no signifi- CD36, an important scavenger receptor for uptake of oxidized cant effect on PMA-induced IL-1b production (Fig. 12A). To low-density lipoprotein, is upregulated in macrophages present in assess whether these receptors are regulated by PKCd during atherosclerotic lesions (4). At the same time, the role of CD36 in IL-1b production, their expression was monitored in rottlerin- innate immunity is speculated from the observation that microglia pretreated and PMA-stimulated THP1 cells. Rottlerin pretreat- and macrophages isolated from CD36-null mice showed marked ment significantly prevented PMA-induced increases in CD11b decrease in fibrillar b-amyloid–induced secretion of cytokines, (Fig. 12B) and TLR2 (Fig. 12C) expression; however, TLR4 chemokines, and reactive oxygen species (48). Therefore, our (Fig. 12D) and CD36 were unaltered (Fig. 12C, Fig. 13). TLR4- present experimental model was relevant to clinical situations and TLR2-specific agonists LPS (Supplemental Fig. 3A)and where monocytes/macrophages exert their deleterious effect by generating an innate immune response and secreting the inflam-

Pam3CSK4 (Supplemental Fig. 3B) also induced secreted IL-1b Downloaded from production in THP1 cells. IRAK1/4 INH, general PKC INH Ro- matory cytokine IL-b (1). 31-8220, and classical PKC INH Go6976 significantly inhibited IRAK pathways are important mediators of innate immune re- both LPS (Supplemental Fig. 3A)- and Pam3CSK4 (Supple- sponse (6–9), and a time-dependent increase in IRAK1 and IRAK4 mental Fig. 3B)-induced IL-1b production. More interestingly, expressions after PMA treatment indicated an induction of this only LPS-induced IL-1b production was unaffected by rottlerin pathway during IL-1b production. IRAK1 expression increased (Supplemental Fig. 3). with time and subsequently a decrease in expression was observed. This can be due to its degradation, a common feature that is asso- http://www.jimmunol.org/ Discussion ciated with the activation of this enzyme (32). A previous report suggested that IRAK1 kinase activity is not important for mediating In the present study we used a human monocytic cell line and IL-1–induced effects that are mediated by IRAK1 (49). However, primary human monocytes for deciphering the role of IRAK1 and this study by using recombinant IRAK1 constructs evaluated the PKCd in IL-1b production. To generate active PKCd, we used importance of IRAK1 kinase activity in effects mediated by IL-1b PMA (22, 23) and a recombinant wild-type PKCd construct in our and not in IL-1b production itself. We observed an early increase in study. Besides enhancing IL-1b production, PMA also induced the kinase activity of this enzyme, which indicated its activation on PMA treatment. Because PMA-induced increases in IL-1b pro- by guest on October 1, 2021 duction were inhibited by the commercially available IRAK1/4 INH, we could conclude that a kinase-active IRAK1/4 is required for IL-1b production. An increase in IRAKM expression was also observed that peaked at 12 h PMA treatment, a time at which ex- pression and activity of IRAK1 had substantially reduced. This late induction of IRAKM seems to negatively counter the positive role of IRAK1 in IL-1b production. At this time point we also saw an inhibition in further increases in IL-1b production, indicating that late induction of IRAKM may inhibit further amplification of in- flammatory response. IRAKM is reported to negatively regulate IRAK1-mediated effects (7, 9). IRAK2 gene silencing was as ef- fective as IRAK1 and IRAK4 in preventing PMA-induced IL-1b production. Previous reports have suggested that IRAK2 can reg- ulate cytokine secretion in both a TLR-dependent and -independent manner (32), and recent reports positioned IRAK2 in TLR signal- ing in a way that is similar to IRAK1 and IRAK4 (14, 15). Silencing IRAKM had no significant effect on IL-1b production, thus in- dicating its dispensable role in PMA-induced IL-1b production. It is known that IRAK1 mediates its effects by activating either the NF-kB or JNK/AP-1 pathway (12). The role of JNK1 and JNK2 in IL-1b production can be deduced from the fact that both the iso- forms of JNK were significantly phosphorylated on PMA treatment FIGURE 13. Model for PKCd- and IRAK1-induced IL-1b production in and their pharmacological inhibition by JNK INH prevented PMA- monocytes. PMA treatment of monocytic cells leads to activation of PKCd. induced IL-1b production. Although a previous report suggested This in turn activates the IRAK1/JNK/AP-1 module to induce IL-1b a role of NF-kB in PMA-induced IL-1b production (50), our results production. Although NF-kB is also activated, its pharmacological in- hibition does not prevent PMA-induced IL-1b production. PKCd-induced rule out this possibility. This can be explained by the fact that all IL-1b production seems to be mediated via TLR2 and CD11b since rot- the three NF-kB DNA-binding motifs present in the IL-1b pro- tlerin prevents their expression and IL-1b production. IRAK2 and IRAK4 moter are located outside the PMA response element (50). In our positively regulate PMA-induced IL-1b production whereas IRAKM has case, significant IL-1b production started much before (1 h) and a negative impact. almost peaked at 6 h. However, significant NF-kB activation was The Journal of Immunology 13 observed only from 12 h. This again indicates the existence of an Existence of such a pathway is suggested from our own obser- NF-kB–independent pathway in IL-1b production. We also ob- vation where significant inhibition in IL-1b production is seen in served significant increases in nuclear AP-1 levels from 30 min of the presence of IL-1R antagonist (data not shown). This antagonist PMA treatment, thus making its role in IL-1b production apparent. is against the Toll-IL-1R domain, which is present in both TLR Regulation of IRAK1 kinase activity by rottlerin, recombinant and IL-1R, indicating that IL-1b production can involve these PKCd, and PKCd siRNA established the positive role of PKCd receptors. A previous report suggested that PKC activators diac- in modulating IRAK1 for IL-1b production. Although the role of ylglycerol and ingenol upregulate CD36 expression in a mouse classical PKC was ruled out in the present study due to lack of macrophage cell line that is inhibited by PKC INH (58). Therefore, inhibition in secreted IL-1b levels by Go6976, other PKCs may at least in THP1 cells CD36 upregulation seems to be independent still be involved. However, an in silico-based phosphorylation site of PKCd since rottlerin pretreatment had no effect on its expression. analysis performed by using a specialized program (51) revealed In the present study, to our knowledge, we report for the first the presence of only a putative high stringency PKCd phosphor- time the role of the IRAK family of proteins in IL-1b production. ylation site in IRAK1 (analysis not shown). All of the other IRAK At the same time, we conclusively demonstrate regulation of isoforms and IRAK1 lacked any high stringency PKC phosphor- IRAK1 by PKCd and the existence of the PKCd/IRAK1/JNK ylation sites. We also observed physical interaction between PKCd axis during PMA-induced IL-1b production. Thus, on the basis of and IRAK1 in quiescent monocytes that was modulated by PMA. present study we propose a novel pathway of IL-1b production The interaction reduced on PMA treatment but was restored in the that involves TLR2, CD11b, and the PKCd/IRAK1/JNK/AP-1 axis. presence of rottlerin. Sometimes due to hyperphosphorylation of the substrate, its dissociation from the upstream kinase is observed

Acknowledgments Downloaded from as reported previously by many workers (24, 52, 53). We thank Dr. Mary Reyland (University of Colorado) for providing PKCd Thus, an endogenous association between IRAK1 and PKCd constructs, and Dr. Madhu Dikshit (Division of Pharmacology, Central confirmed the regulation of IRAK1 by rottlerin. Whether PKCd Drug Research Institute) for critical inputs. We gratefully acknowledge regulates IRAK1 kinase activity by directly phosphorylating this the technical help provided by C.P. Pandey, M. Chaturvedi, and A.L. isoform needs further investigation. Vishwakarma. We also used various combinations of PKCd, IRAK1/4, and JNK inhibitors so as to ascertain whether these proteins also feed Disclosures http://www.jimmunol.org/ into other pathways for IL-1b production. We achieved ∼76, 78, The authors have no financial conflicts of interest. and 84% inhibition in PMA-induced IL-1b production with rot- tlerin, IRAK1/4, or JNK INH, respectively. Thus, it can be said References that these proteins are involved in a major way in PMA-induced 1. Maedler, K., G. Dharmadhikari, D. M. Schumann, and J. Størling. 2009. In- IL-1b production. Similar levels of inhibitions also indicate terleukin-1b targeted therapy for type 2 diabetes. Expert Opin. Biol. Ther. 9: working of the PKCd/IRAK1/JNK axis during IL-1b production. 1177–1188. Maximum inhibition was observed when all the three inhibitors 2. Schroder, K., R. Zhou, and J. Tschopp. 2010. The NLRP3 inflammasome: a sensor for metabolic danger? Science 327: 296–300. were used together, indicating that these proteins may regulate

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