PKCδ-IRAK1 axis regulates oxidized LDL-induced IL-1β production in monocytes.

This study examined the role of interleukin (IL)-1 receptor-associated kinase (IRAK) and protein kinase C (PKC) in oxidized LDL (Ox-LDL)-induced monocyte IL-1β production. In THP1 cells, Ox-LDL induced time-dependent secretory IL-1β and IRAK1 activity; IRAK4, IRAK3, and CD36 protein expression; PKCδ-JNK1 phosphorylation; and AP-1 activation. IRAK1/4 siRNA and inhibitor (INH)-attenuated Ox-LDL induced secreted IL-1β and pro-IL-1β mRNA and pro-IL-1β and mature IL-1β protein expression, respectively. Diphenyleneiodonium chloride (NADPH oxidase INH) and N-acetylcysteine (free radical scavenger) attenuated Ox-LDL-induced reactive oxygen species generation, caspase-1 activity, and pro-IL-1β and mature IL-1β expression. Ox-LDL-induced secretory IL-1β production was abrogated in the presence of JNK INH II, Tanshinone IIa, Ro-31-8220, Go6976, Rottlerin, and PKCδ siRNA. PKCδ siRNA attenuated the Ox-LDL-induced increase in IRAK1 kinase activity, JNK1 phosphorylation, and AP-1 activation. In THP1 macrophages, CD36, toll-like receptor (TLR)2, TLR4, TLR6, and PKCδ siRNA prevented Ox-LDL-induced PKCδ and IRAK1 activation and IL-1β production. Enhanced Ox-LDL and IL-1β in systemic inflammatory response syndrome (SIRS) patient plasma demonstrated positive correlation with each other and with disease severity scores. Ox-LDL-containing plasma induced PKCδ and IRAK1 phosphorylation and IL-1β production in a CD36-, TLR2-, TLR4-, and TLR6-dependent manner in primary human monocytes. Results suggest involvement of CD36, TLR2, TLR4, TLR6, and the PKCδ-IRAK1-JNK1-AP-1 axis in Ox-LDL-induced IL-1β production.

Oxidized LDL (Ox-LDL) in various acute or chronic infl ammatory diseases can be an independent risk factor for cardiovascular complications ( 1,2 ). Ox-LDL itself serves as a pro-infl ammatory molecule and contributes to the generation of various infl ammatory cytokines ( 3,4 ). Elevated Ox-LDL and infl ammatory response were observed in extremely obese pediatric subjects ( 5 ). Recent reports suggest that Ox-LDL can induce sterile infl ammation by stimulating production of various infl ammatory cytokines, including interleukin (IL)-1 ␤ ( 6, 7 ). Sterile infl ammation is characterized by the recruitment of neutrophils and macrophages and production of infl ammatory cytokines like IL-1 ␤ and TNF-␣ ( 8 ). Several exogenous agents like asbestos and silica, and endogenous stimuli like RNA, DNA, and cytokines can induce sterile infl ammation ( 8 ). In monocytic cells, Ox-LDL-induced sterile infl ammation was dependent on CD36-induced heterodimerization of toll-like receptor (TLR)4 and TLR6 ( 6 ). Binding of Ox-LDL to CD36 was found to be the initial step that was important for TLR heterodimerization and induction of sterile infl ammatory response ( 6 ). IL-1 ␤ -induced sterile infl ammation is also reported during acute pancreatitis ( 9 ). In addition, IL-␤ has been shown to induce sterile infl ammation by regulating macrophage migration ( 10 ). Moreover, evidence for IL-␤ -induced sterile infl ammation also comes from studies in which mice were subjected to sterile injuries ( 11 ).
Traumatic injury often induces a sterile systemic infl ammatory response syndrome (SIRS) in humans, and involves purchased from Santa Cruz Biotechnology, Inc. and Dharmacon (Chicago, IL). Anti-CD36 (FA6-152) antibody was procured from Abcam (Cambridge, MA). ECL reagent was from GE Healthcare (USA). Tissue culture reagents were procured from Invitrogen (USA). All other fi ne chemicals used in the study were procured from Sigma.

Study population
In the present study, 74 healthy volunteers and 41 SIRS patients were recruited and evaluated for circulating Ox-LDL and plasma IL-1 ␤ . Ethical approval was taken from the institutional ethics committee (human research) of CSIR-Central Drug Research Institute, King George's Medical University, and Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, and written consent was obtained from the patients' surrogates. Kin, caretakers, or guardians consented on the behalf of participants whose capacity to consent was reduced and the institutional committee approved this consent procedure. Ethical guidelines were in agreement with the Declaration of Helsinki. Critically ill patients were admitted to the intensive care unit of King George's Medical University, Lucknow, and SIRS was diagnosed by the presence of two or more of the following criteria: temperature >38°C or <36°C; heart rate >90 beats/min; respiratory rate >20 breaths/min or PaCO 2 <32 mm Hg; and an alteration in the white blood cell count >12,000 cells/ l. Inclusion criteria for the patients enrolled in the present study were patients of trauma, postoperative surgical patients, and patients with respiratory illness (COPD, asthma). The exclusion criteria for SIRS patients were patients older than 80 years, cardiac failure (class III or IV), liver insuffi ciency, and the presence of HIV, HBV, HCV, infection, or cancer. Disease severity index [Acute Physiology and Chronic Health Evaluation (APACHE) II scores and Sequential Organ System Failure Assessment (SOFA) scores] ( 26 ), along with other clinical pathology tests, were monitored at the time of admission in the intensive care unit. Among the SIRS patients, 68% were men and 32% were women, with a mean ± standard error (SE) age of 44 ± 5 years ( Table 1 ). The mean ± SE age of healthy subjects was 42 ± 4 years, out of which 75% were male participants and 25% were female participants ( Table 1 ). Blood samples were collected in tubes containing 3.8% tri-sodium citrate (9:1 ratio) from healthy subjects and SIRS patients with the help of a central venous catheter, and plasma was separated after centrifugation at 13,000 g for 7 min ( 27 ). Plasma was used immediately or stored at Ϫ 70°C for assessment of circulating Ox-LDL level and plasma IL-1 ␤ . Repeated freezing and thawing of samples were avoided to prevent degradation of plasma Ox-LDL and IL-1 ␤ levels .

Circulating Ox-LDL measurement
Circulating Ox-LDL was measured using an Ox-LDL competitive ELISA kit (Mercodia AB, Uppsala, Sweden). As per manufacture's protocol, plasma samples were initially diluted with sample buffer. Calibrator (25 l), control and diluted samples, along with 100 l of assay buffer were added into appropriate wells precoated activation of the innate immune response ( 12 ). The severity of immune response is often associated with the amount of circulating cytokines present in the patient ( 12,13 ). Incidence of multiple organ failure and mortality increases with the increasing infl ammatory load ( 12 ). TLR2/4 expression on peripheral blood mononuclear cell , as well as serum TNF-␣ , IL-␤ , and IL-8, was signifi cantly higher in SIRS patients ( 14 ).
Exposure of monocytic cells to organic dust leads to production of several inflammatory cytokines, like TNF-␣ and IL-6, which seems to be mediated by protein kinase C (PKC) ␦ , along with some other PKC isoforms ( 15 ). PKC ␦ is a serine-threonine protein kinase that can be activated by calcium and diacylglycerol and plays an important role in infl ammation (16)(17)(18). It has been shown to be involved in sepsis ( 19,20 ) and seems to mediate sepsis-induced lung injury ( 19 ). PKC ␦ also mediates high glucose-induced activation of the TLR pathway and production of infl ammatory cytokines in monocytic cells ( 21 ). Interestingly, asbestos-induced peribronchiolar cell proliferation and cytokine production are attenuated in the lungs of protein kinase C ␦ knockout mice ( 22 ).
The IL-1 receptor (IL-1R)-associated kinase (IRAK) family of kinases represents important mediators of innate immunity and plays a crucial role in the signaling cascade induced by the TLR/IL-1R family ( 17,(23)(24)(25). The IRAK family consists of four members, namely IRAK1, IRAK2, IRAK3 (IRAKM), and IRAK4. IRAK1, IRAK2, and IRAK4 positively regulate the immune response, and IRAK3 usually antagonizes their effect by disrupting the IRAK1/TNFRassociated factor 6 complex ( 17,23,25 ). Out of all of these kinases, IRAK1 and IRAK4 are widely studied proteins and have been proposed to be true kinases, but their kinase activity is still under investigation ( 17,25 ).
In the present study, we hypothesized that Ox-LDL can modulate the PKC and IRAK pathways in monocytic cells to induce sterile infl ammation by stimulating IL-␤ production . The present study demonstrates the role of PKC ␦ in Ox-LDL-induced sterile infl ammation by directly activating the IRAK1-JNK axis for IL-1 ␤ production. This hypothesis has clinical relevance because high Ox-LDL plasma in SIRS individuals primes monocytes for IL-␤ overproduction by activating the PKC ␦ -IRAK1 axis.

Assay for secretory IL-1 ␤ production
The production of IL-1 ␤ after treatment with Ox-LDL, different pathway INHs and antibodies was measured in the media by conventional ELISA (BD OptEIA TM set human IL-1 ␤ ; BD Biosciences, San Diego, CA) as described earlier ( 17 ). In brief, supernatants were collected from control and treated monocytic cells and incubated for 2 h at room temperature in overnight capture antibodycoated ELISA plates . After incubation, wells were washed with PBS containing 0.05% Tween-20 and incubated with detection antibody followed by washing and enzyme reagent incubation. The color was developed by adding 3,3',5,5'-tetramethylbenzidine substrate reagent set (BD Biosciences) and subsequently read at 450 nm and 570 nm on an ELISA plate reader (BioTek Instruments, Inc.). Standard IL-1 ␤ provided in the kit was used for drawing the standard and calculation of absolute IL-1 ␤ levels.

Immunoprecipitation and in vitro kinase assay
Cells from different experimental groups were lysed in 0.1% Nonidet P-40 lysis buffer [50 mM Tris-Cl (pH 8.0), 137 mM sodium chloride, 2 mM EDTA, 5% glycerol, and 0.1% Nonidet P-40] supplemented with 1:100 protease INH cocktail. The lysates were centrifuged at 15,500 g , supernatants were collected, and protein concentration was measured. Pre-clearing of cell lysates was performed by incubating 400 g of cell extracts from different experimental groups with 20 l protein A Sepharose beads (50% slurry) for 45 min at 4°C. After centrifugation at 14,000 g for 10 min, the supernatant was mixed with 2.0 g/ml rabbit anti-IRAK1 antibody and incubated at 4°C overnight. Subsequently, 20 l of protein A Sepharose beads (50% slurry) was mixed and further rotated for 2 h at 4°C. The protein A Sepharose beads were spun down and washed four times with lysis buffer and two times with 0.1M LiCl. The immunoprecipitates were processed for immunoblotting as desired.
with anti-Ox-LDL monoclonal antibody. Plates were incubated on a plate shaker (700-900 rpm) for 2 h at room temperature (23-25°C). After rinsing with wash buffer, 100 l of enzyme conjugate was added to each well and incubated for 1 h at room temperature. After subsequent washing, 3,3',5,5'-tetramethylbenzidine substrate was added and the developed color was measured using an ELISA reader (BioTek Instruments, Inc., USA) at a wavelength of 450 nm ( 28 ). Standard curve was prepared for each assay run using calibrators and control supplied along with the assay kit. Cu 2+modifi ed LDL (50-500 ng/ml) was used as standard solution ( 4 ) to quantify the circulating plasma Ox-LDL in micrograms per milliliter for the treatment in primary monocytes isolated from healthy volunteers.

Human monocyte isolation, THP1 cell culture, and treatments
Primary human monocytes were isolated, as described earlier with slight modifi cation ( 17,29 ), from healthy donors after their informed consent . Whole blood was centrifuged at 250 g for 20 min and the upper layer (rich in platelets) plateletrich plasma (PRP ) was removed. The remaining blood was centrifuged at 650 g for 20 min and the buffy coat was collected. It was mixed with saline and subjected to dextran sedimentation. The upper layer (rich in leukocytes) was collected and centrifuged at 500 g for 5 min at room temperature. Pellets were resuspended in HBSS containing glucose. Density gradient centrifugation utilizing Percoll 1080 and 1065 was done at 700 g for 15 min and the interface layer was collected and washed with glucose HBSS. The pellet was resuspended in RPMI-1640, loaded on hyper-osmotic gradient, and the interface layer of monocytes was adhered in RPMI-1640 containing 10% FBS for 1 h and subsequently used for experiments ( 17,29 ). Viability of cells was found to be >95%, as assessed by trypan blue staining, and purity of cells was found to be >95%, as assessed by CD14 + cells by fl ow cytometry. In addition to this human monocytic cell line, THP1 was cultured in RPMI-1640 containing 10% heat-inactivated FBS, 100 IU/ml penicillin, and 100 g/ml streptomycin. Primary monocytes were preincubated with CD36 antibody (5 g/ml) or with respective isotype and vehicle control for 1 h. Subsequently, monocytes were treated with 40% (v/v) plasma ( 4 ) from healthy subjects with low (6.7 ± 0.3 g/ml) and high (26.5 ± 0.5 g/ml) Ox-LDL and plasma from SIRS patients with low (12 ± 0.07 g/ml) and high (32 ± 2 g/ml) Ox-LDL ( 4 ). After respective treatments, supernatant was collected for IL-1 ␤ measurement and cell lysates were prepared for Western blotting.

Isolation, purifi cation, and characterization of Ox-LDL
LDL (d = 1.019-1.063 g/ml) was isolated from the plasma of healthy volunteers by sequential ultracentrifugation ( 31 ). Ox-LDL was prepared by dialyzing the LDL in PBS overnight at 4°C. LDL protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, IL). Native LDL (0.2 mg/ml) diluted in PBS was oxidized by exposure to 5 M CuSO 4 in PBS at 37°C for 24 h. The oxidation was terminated by addition of Na 2 EDTA

Caspase-1 fl uorometric assay
Caspase-1 activity was assayed by using a caspase-1 fl uorometric assay kit (R&D Systems, Inc., Minneapolis, MN). After various treatments, cells were collected by centrifugation at 250 g for 10 min. The kit buffer was used for cell lysis. The supernatant obtained after centrifugation at 10,000 g was used for caspase-1 assay. Total protein (200 g) was mixed with an equal volume of 2× reaction buffer in a microplate. Reactions were initiated by the addition 5 l of caspase-1 fl uorogenic substrate (WEHD-AFC). The reaction was carried out at 37°C for 2 h. Plates were read at excitation 400 nm and emission 505 nm in an LS 55 fl uorescence plate reader (Perkin Elmer, Waltham, MA). The results were expressed as fold increase in caspase-1 activity of induced cells over that of noninduced cells ( 34 ).

Expression of IL-1 ␤ by real-time PCR
Total RNA from THP1 cells was extracted by using TRI reagent. For quantitative (q)RT-PCR analysis of IL-1 ␤ , cDNA was synthesized from 1 g of RNA by using a commercially available cDNA synthesis kit (Fermentas RevertAid fi rst strand DNA synthesis kit, Lithuania ). Real-time PCR was done in a 25 l reaction by using Maxima® CYBR Green/ROX qPCR Master Mix (2×) (Fermentas Life Sciences, Lithuania), IL-1 ␤ (forward primer-CTCTCTCACCTCTCCTACTCAC, reverse primer-ACACTGCTACTTCTTGCCCC), actin (forward primer-AACTGGAACGGTGAAGGTG, reverse primer-CTGTGTG-GACTTGGGAGAGG) specifi c primers, and LightCycler® 480 realtime PCR system (Roche Applied Science, Mannheim, Germany ). Three step cycling protocol (initial denaturation at 95°C for 10 min, 35 cycles of 15 s denaturation at 95°C, 30 s annealing at 60°C, and 30 s extension at 72°C) was used to amplify the genes ( 35,36 ). Relative fold difference between an experimental and calibrator sample was calculated by using comparative Ct (2 Ϫ ⌬ ⌬ Ct ) method. Actin was used as internal standard to calculate the relative expression ( 37 ).

Statistical analysis
Results are expressed as the mean ± SE. The data obtained from control and SIRS patient samples were analyzed by Kolmogorov-Smirnov test for normal distribution. The Pearson product-moment correlation coeffi cient ( r ) was used to establish the association of the two variables. Unpaired Student's t -test was used to calculate the signifi cant difference between two groups. The signifi cance of difference between the means of three or more groups was determined by one-way ANOVA followed by Tukey-Kramer post hoc multiple comparison test. P р 0.05 was considered statistically signifi cant. Blots represent one of three or more similar experiments. All statistical analyses were performed with the GraphPad Prism 5.0 program (GraphPad Inc., San Diego, CA).

Ox-LDL induces IL-1 ␤ production and activation of IRAK pathway
THP1 monocytic cells were treated with Ox-LDL (40 g/ml) for the indicated time points and secretory IRAK1 kinase assay was performed as described earlier ( 17 ). Briefl y, the immune complexes were washed with kinase assay buffer [20 mM MOPS (pH 7.2), 50 mM MgCl 2 , 2 mM EGTA, and 1 mM dithiothreitol). The reaction was carried out in the presence of 5 g of MBP substrate, 0.5 mM ATP, and 10 Ci of [ ␥ 32 -P] ATP for 30 min at 30°C ( 17 ). Reactions were stopped by the addition of 15 l of 6× SDS-PAGE sample buffers and subsequently boiled. Supernatants were subjected to SDS-PAGE and transferred to PVDF membranes. Phosphorylation of the substrate was measured by autoradiography.

AP-1 activity assay
AP-1 activity was measured at different time points of Ox-LDL treatment by using a commercially available ELISA kit (TransAM TM AP-1-c-Jun, Active Motif Co., Ltd., Carlsbad, CA). Nuclear extracts were prepared as per the instructions in the kits. Briefl y, after treatment, monocytic cells were collected and washed with ice-cold phosphatase INH buffer (125 mM NaF, 250 mM ␤glycerophosphate, 250 mM para-nitrophenyl phosphate, and 25 mM NaVO 3 ) and resuspended in 1 ml of ice-cold hypotonic buffer [20 mM HEPES (pH 7.5), 5 mM NaF, 10 M Na 2 MoO 4 , and 0.1 mM EDTA). The cells were allowed to swell for 15 min on ice. Fifty 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 the supernatant (cytoplasmic fraction) was removed. The nuclear pellet was suspended in 50 l of complete lysis buffer for 30 min on a rocking platform. The lysate was centrifuged at 15,000 g at 4°C for 10 min and the nuclear extract was used for AP-1 (c-jun ) assay after protein quantifi cation. AP-1 was measured by loading 10 g of nuclear extract onto a well of a 96-well microtiter plate coated with oligonucleotide 5 ′ -TGAGTCA-3 ′ for 1 h. After washing three times, monoclonal antibody against c-jun was added to the appropriate wells and incubated further for 1 h at room temperature. Anti-IgG HRP conjugate, in a volume of 100 l, was then added and further incubated for 1 h at 25°C. Absorbance at 450 nm was measured after the addition of tetramethylbenzene solution. Absolute levels of the transcription factor were quantifi ed by setting up standard curves with the help of reagents provided in the kit.

siRNA transfection
Transfections were performed by using an Amaxa Nucleofector machine (Amaxa, Cologne, Germany), as described earlier ( 17 ), and in the optimized protocol for THP1 and primary monocytes as provided by the manufacturer. Briefl y, 1 × 10 6 cells in 100 l transfection reagent provided in the kit (Cell Line Nucleofector kit V) were transfected with 3.0 g of control, IRAK1, IRAK2, IRAK3, IRAK4, TLR2, TLR4, TLR6, CD36, or PKC ␦ siRNA. Nucleofector machine program V001 was used for THP1 and Y001 for primary monocytes. After transfection, cells were removed in 0.5 ml RPMI and plated in 1 ml of prewarmed medium in 6-well plates. THP1 macrophages were transfected with control, PKC ␦ , TLR2, TLR4, TLR6, or CD36 siRNA using Lipofectamine 2000 transfection reagent according to the manufacturer's instructions. Briefl y, THP1 cells were differentiated with PMA (100 nM) for 24 h. Lipofectamine and siRNA (3 g) were incubated together at room temperature for 20 min and the complex formed was added to the cells.
After 18 h of transfection, Ox-LDL treatment was given for 15 min to measure PKC ␦ and IRAK1 phosphorylation in THP1 cells, primary monocytes, and THP1 macrophages. CD36 expression was also measured in THP1 macrophages. Secretory IL-1 ␤ was measured after 48 h of Ox-LDL treatment. Expression of recombinant green fl uorescent protein (provided in the kit) and FITC-labeled control siRNA were used as markers for monitoring the transfection efficiency. Gene silencing was measured by Western blotting. any further increase at later time points ( Fig. 1B ). Furthermore, we also observed increased IRAK3 expression in a time-dependent manner up to 72 h of Ox-LDL stimulation, but no change was found in expression of IRAK2 ( Fig. 1B, C ). No difference in expression of IRAK2 was observed after Ox-LDL stimulation. Expression of IRAK3 was increased in a time-dependent manner up to 72 h of Ox-LDL stimulation ( Fig. 1B, C ). Expression of IRAK4 was also signifi cantly increased at 30 min of Ox-LDL stimulation, and this was maximum at 24 h. A decrease in IRAK4 expression was observed at 48 and 72 h of Ox-LDL stimulation, but this was still signifi cantly more than the control levels ( Fig. 1B, C ). Because it is reported that IRAK1 is downstream to IRAK4 and relays the signal forward ( 25 ), we performed IRAK1 kinase assay to ascertain the activation of the IRAK4-IRAK1 signaling pathway. A signifi cant IL-1 ␤ was measured in the supernatant ( Fig. 1A ). A timedependent increase in IL-1 ␤ production was observed after Ox-LDL treatment ( Fig. 1A ). The treatment with Ox-LDL for 6 h signifi cantly increased secreted IL-1 ␤ ( ‫ف‬ 4-fold), and this was further increased with time reaching maximum at 48 h ( ‫ف‬ 25-fold). At 72 h, the secreted IL-1 ␤ was not significantly different from that observed at 48 h ( Fig. 1A ). However, LDL (40 g/ml) treatment for 72 h had no effect on IL-1 ␤ production ( Fig. 1A ). Because the IRAK family of proteins mediates innate immune response generated by the TLR/IL-1R receptor ( 39 ), activation of different IRAK proteins was studied. We monitored time-dependent expression of all IRAK isoforms up to 72 h in THP1 monocytes, after Ox-LDL treatment ( Fig. 1B, C ). A moderate but signifi cant increase in expression of IRAK1 was observed after 15 and 30 min of Ox-LDL stimulation without  determine the role of each IRAK isoform in Ox-LDLinduced IL-1 ␤ production, isoform-specifi c siRNAs were used. A signifi cant reduction in IRAK1, -2, -3, and -4 expression was observed on treatment with their specifi c siRNA ( Fig. 2B-E ). IRAK1-and IRAK4-specifi c siRNA signifi cantly inhibited Ox-LDL-induced secretory IL-1 ␤ , while no change was observed with IRAK2 and IRAK3 siRNA ( Fig. 2B-E ).

IRAK1/4 regulates Ox-LDL-induced IL-1 ␤ transcription
Because IL-1 ␤ production is regulated at multiple levels, including gene transcription, translation, and processing, expression of IL-1 ␤ at mRNA level was measured by real-time RT-PCR and at protein level by Western blotting.
increase in kinase activity of IRAK1 was observed after 15 min ( ‫ف‬ 2-fold) and 30 min ( ‫ف‬ 4-fold) of Ox-LDL treatment, which diminished at later time points ( Fig. 1D ), indicating activation of the TLR/IL1-R signaling pathway. A time-dependent increase in CD36 protein expression was also observed after Ox-LDL treatment (supplementary Fig. I).

Involvement of the JNK1-AP-1 axis in Ox-LDL-induced IL-1 ␤ production
Because downstream signaling of IRAK involves the JNK pathway ( 40 ), we performed phospho-JNK blotting in THP1 lysates obtained after Ox-LDL stimulation for different time points . An initial activation of JNK1 ( ‫ف‬ 2 to 4-fold) at 15 and 30 min of Ox-LDL treatment was observed, which subsided at later time points ( Fig. 4A ). Interestingly, specifi c activation of JNK1 was observed, but there was no signifi cant increase in JNK2 phosphorylation after Ox-LDL treatment ( Fig. 4A ). Because further downstream signaling of JNK involves AP-1-induced gene transcription, we therefore evaluated nuclear AP-1 DNA binding activity by using a TransAM TM AP-1 c-Jun ELISA kit ( Fig. 4B ). Ox-LDL classical (G06976) PKC INHs. The Ro-31-8220 and Go-6976 signifi cantly reduced Ox-LDL-induced secretory IL-1 ␤ production ( Fig. 5A ). More importantly, PKC ␦ -specifi c INH Rottlerin also signifi cantly reduced Ox-LDL-induced IL-1 ␤ production ( Fig. 5A ). Previous studies have also suggested a role of PKC ␦ in IL-1 ␤ production from monocytes ( 18 ). On expected lines, we did see a time-dependent activation of PKC ␦ after Ox-LDL treatment ( Fig. 5B ). PKC ␦ activation was observed starting from 15 min up to 72 h and activation was maximum ( ‫ف‬ 5-fold) at 12 h, confi rming that Ox-LDL treatment activates PKC ␦ ( Fig. 5B ).

PKC ␦ mediates Ox-LDL-induced IRAK1 activation and IL-1 ␤ production
Previous reports suggest a crucial role of PKC in IL-1 ␤ production from monocytes ( 18 ). To evaluate the role of various PKC isoforms in secretory IL-1 ␤ production, experiments were carried out in the presence of different classes of PKC INHs ( Fig. 5A ). Ox-LDL-induced IL-1 ␤ production was measured in the presence of general (Ro-31-8220) and experiments were performed to test whether the PKC ␦ -IRAK pathway feeds into the JNK-AP-1 axis during Ox-LDLinduced IL-1 ␤ production. Ox-LDL-induced JNK-AP-1 axis activation was evaluated in the presence of Rottlerin and IRAK1/4 INH. JNK activation was monitored at 15 min of Ox-LDL stimulation after pretreatment with Rottlerin, IRAK1/4 INH ( Fig. 6A ), and PKC ␦ siRNA ( Fig. 6B ). Significant inhibition in JNK phosphorylation was observed in the presence of these INHs and PKC ␦ siRNA, thus indicating that the PKC ␦ -IRAK1 axis feeds into the JNK pathway. Because AP-1 inhibition by Tanshinone IIa also inhibits Ox-LDL-induced IL-1 ␤ production, we determined the AP-1 level at 30 min of Ox-LDL stimulation in Rottlerin, IRAK1/4 INH, JNK INH II, and PKC ␦ siRNA pretreated THP1 cells ( Fig. 6C ). Signifi cant inhibition in Ox-LDL-induced AP-1 activity by these INHs and PKC ␦ siRNA indicates that PKC ␦induced IL-1 ␤ production involves the PKC ␦ -IRAK1-JNK-AP-1 axis. In primary human monocytes also, Ox-LDL induced time-dependent PKC ␦ phosphorylation ( Fig. 6D ).
Further, we explored the role of CD36 and TLRs in Ox-LDL-induced PKC ␦ and IRAK1 activation and IL-1 ␤ production in THP1 monocyte-derived macrophages. Ox-LDL enhanced PKC ␦ phosphorylation ( from as early as 5 min of Ox-LDL treatment, and a significant increase was observed from 15 min of treatment. The increase in PKC ␦ phosphorylation was sustained until the last point of analysis. Ox-LDL signifi cantly enhanced IL-1 ␤ production in primary human monocytes, while LDL had no signifi cant effect ( Fig. 6E ). Rottlerin ( Fig. 6E ) and PKC ␦ siRNA ( Fig. 6F ) pretreatment signifi cantly attenuated Ox-LDL-induced IL-1 ␤ production in these cells as well ( Fig. 6E and Fig. 6F , respectively).

Role of CD36 and TLR in Ox-LDL-induced IL-1 ␤ production
To explore the involvement of CD36 and TLRs in Ox-LDL-induced IL-1 ␤ production, THP1 cells were pretreated with TLR6, TLR4, TLR2, and CD36 siRNA, and subsequently stimulated with Ox-LDL.

Elevated Ox-LDL and IL-1 ␤ in SIRS patients
Ox-LDL and IL-1 ␤ were measured in the plasma of healthy subjects (n = 74) and SIRS patients (n = 41). Patient demographic characteristics including heart rate, Fig. 8. Plasma Ox-LDL and IL-1 ␤ augmented in SIRS patients. Plasma analysis was done in healthy and SIRS individuals. Bar diagrams representing Ox-LDL (A) and IL-1 ␤ (B). To analyze the association between Ox-LDL and IL-1 ␤ , the Pearson product-moment correlation coeffi cient ( r ) was used. Line graphs represent correlation between Ox-LDL and IL-1 ␤ in healthy subjects (n = 74) (C) and SIRS patients (n = 41) (D). E: Bar graph representing Ox-LDL-LDL ratio in healthy and SIRS individuals. F: Line graph representing correlation between Ox-LDL-LDL ratio and IL-1 ␤ in SIRS patients. Values represent mean ± SE. * P < 0.05, *** P < 0.001 versus healthy subjects. respectively] and high [26.5 ± 0.5 g/ml (control) and 32 ± 2 g/ml (SIRS), respectively] amounts of Ox-LDL with or without Ox-LDL receptor CD36 FA6 antibody and its isotype control.
To determine a correlation between circulating Ox-LDL, IL-1 ␤ , and disease severity scores (SOFA and APACHE II), we applied the Pearson correlation coeffi cient, which showed a positive correlation between an increase in circulating Ox-LDL and disease severity score SOFA ( r = 0.7, P < 0.0001; Fig. 9A ) and APACHE II ( r = 0.57, P < 0.0001; Fig. 9B ) in SIRS patients. Similarly, a positive correlation was observed between plasma IL-1 ␤ and SOFA ( r = 0.5, P = 0.0008; Fig. 9C ) and APACHE II ( r = 0.52, P = 0.0004; Fig. 9D ) scores in SIRS patients, indicating an increase in Ox-LDL and IL-1 ␤ with disease severity.

SIRS plasma with enhanced Ox-LDL primes monocytes for PKC ␦ -IRAK1 hyper-phosphorylation and IL-1 ␤ overproduction
Because IL-1 ␤ and Ox-LDL increase showed a positive correlation in both healthy subjects and SIRS patients, and PKC ␦ and IRAK are known to modulate IL-1 ␤ production from human monocytes ( 17,18 ), the dose-dependent effect of Ox-LDL on phospho-PKC ␦ , phospho-IRAK1, and IL-1 ␤ was monitored by treating primary monocytes with control and SIRS plasma containing low and high levels of Ox-LDL.
Monocytes from healthy volunteers were treated with 40% plasma (v/v) from healthy or SIRS patients containing low [6.7 ± 0.3 g/ml (control) and 12 ± 0.07 g/ml (SIRS), events during Ox-LDL-induced IL-1 ␤ production . Because Ox-LDL treatment induces infl ammation and subsequent cholesterol accumulation causes cell death ( 41,42 ), we have used an optimal concentration that induces signifi cant IL-1 ␤ production along with minimal cell death; this has been routinely used by other investigators as well ( 6,30 ).
A time-dependent increase in Ox-LDL-induced IL-1 ␤ production and IRAK1 kinase activity indicated that there is a positive correlation between the two. IRAK1 activation preceded a signifi cant increase in IL-1 ␤ production, thus indicating its role in the production of the infl ammatory cytokine. A signifi cant increase in IRAK4 expression was also observed, thus confi rming the induction of IRAK1 and IRAK4 during Ox-LDL-induced IL-1 ␤ production. A time-dependent increase in IRAK3 can explain the saturating levels of IL-1 ␤ observed at later time points. IRAK3 can negatively regulate a positive infl ammatory response ( 43 ).

DISCUSSION
In the present study, we have evaluated the role of the PKC and IRAK kinase families and associated signaling Fig. 10. Control plasma with low and high Ox-LDL induces PKC ␦ and IRAK1 activation and IL-1 ␤ production in monocytes. Human primary monocytes were pretreated for 1 h with or without CD36 antibody FA6 and isotype control antibody (5 g/ml), and then plasma from healthy subjects containing low Ox-LDL or high Ox-LDL was added. Total and phosphorylated PKC ␦ (A) and IRAK1 (B) were measured after 15 min of stimulation by immunoblotting with phospho-PKC ␦ and phospho-IRAK1 antibody, respectively (n = 3). C: IL-1 ␤ level was measured in the supernatant after 48 h treatment of plasma derived from healthy subjects (in triplicate, n = 5). Blots represent one of three similar experiments. Values represent mean ± SE. * P < 0.05, *** P < 0.001 versus control; @@ P < 0.01, @@@ P < 0.001 low Ox-LDL versus high Ox-LDL; ### P < 0.001 high Ox-LDL versus high Ox-LDL + FA6.
pro-IL-1 ␤ and mature IL-1 ␤ protein expression were signifi cantly attenuated by free radical scavenger NAC and NADPH oxidase INH DPI, it is quite possible that ROS play a role in IL-1 ␤ processing. Earlier studies have also shown that free radicals mediated caspase-1 activation and IL-1 ␤ production in THP1 monocytes and macrophages after Ox-LDL stimulation ( 7,46 ).
Upstream positioning of IRAK in the JNK pathway has been done earlier ( 25 ), and in the present study also, the IRAK1/4 INH signifi cantly attenuated Ox-LDL-induced JNK1 phosphorylation. Previous reports suggest a role of the JNK-AP-1 axis in IL-1 ␤ production ( 17 ). Ox-LDL seems to induce JNK1-specifi c IL-1 ␤ production because JNK2 phosphorylation was unaffected by Ox-LDL treatment. A simultaneous increase in Ox-LDL-induced AP-1 activity indicates that JNK1-mediated effects are transduced through AP-1. Because Ox-LDL-induced IL-1 ␤ production was signifi cantly attenuated in the presence of the JNK-specifi c and AP-1 INHs, it can be speculated that the JNK1-AP-1 IL-1 ␤ production can be regulated at several levels, including transcription, translation, processing, and secretion ( 44 ). In the present study, Ox-LDL induces IRAK1-dependent IL-1 ␤ transcription because signifi cant inhibition in pro-IL-1 ␤ transcription was observed with IRAK1/4 INH. Accumulation of cholesterol crystals in the monocytes is known to increase the caspase-1 activity via activating the NLRP3 infl ammasome ( 45 ). Recently, it was shown in macrophages that Ox-LDL induces IL-1 ␤ production by stimulating IL-1 ␤ transcription and also processing by activating the NLRP3 infl ammasome-caspase-1 pathway ( 7 ). However, in the present study, caspase-1 activation seems to be independent of the IRAK pathway. This can be explained by the fact that production of IL-1 ␤ involves two steps: 1 ) TLR-induced transcription of IL-1 ␤ to form pro-IL-1 ␤ ; and 2 ) infl ammasome-induced activation of caspase-1, which then processes the pro-IL-1 ␤ to form the mature IL-1 ␤ . In the present study, Ox-LDL induced ROS generation and caspase-1 activity. Because Ox-LDL-induced Fig. 11. SIRS plasma with low and high Ox-LDL induces CD36-dependent PKC ␦ and IRAK1 activation and IL-1 ␤ production in monocytes. Primary human monocytes were pretreated for 1 h with CD36 antibody FA6 (5 g/ml) and then plasma derived from SIRS patients containing low Ox-LDL or high Ox-LDL was added. Subsequently, total and phosphorylated PKC ␦ (A) and IRAK1 (B) were monitored after 15 min of stimulation (n = 3); IL-1 ␤ in the supernatant was measured after 48 h of treatment (C) (in triplicate, n = 5). Values represent mean ± SE. * P < 0.05, *** P < 0.001 versus control; @@@ P < 0.001 low Ox-LDL versus high Ox-LDL; # P < 0.05, ### P < 0.001 high Ox-LDL versus high Ox-LDL + FA6. axis because PKC ␦ SiRNA prevented Ox-LDL-induced IRAK1-JNK-AP-1 activation and IL-1 ␤ production.
There are several ways by which PKC ␦ can be activated during IL-1 ␤ production, including direct activation by Src family kinases ( 48,49 ), which are known to associate with CD36 during Ox-LDL-induced macrophage foam cell formation ( 50 ). Several studies implicate PKC ␦ in TLR-induced cytokine production ( 18,51 ). PKC ␦ can bind to Toll-interleukin 1 receptor (TIR) domain-containing adapter protein/MyD88 adapter-like (Mal) , an adaptor protein for TLR2 and TLR4, and promote TLR signaling ( 52 ). A recent report also suggests a role of PKC ␦ in macrophage foam cell formation by regulating expression of SRA and CD36 ( 53 ). Our present fi ndings have immense implications for disorders like atherosclerosis, where PKC ␦ can act as a double-edged sword by preventing both Ox-LDL-induced infl ammation and macrophage foam cell formation. axis mediates Ox-LDL-induced IL-1 ␤ transcription. Earlier reports also demonstrate the role of AP-1 in IL-1 ␤ transcription ( 47 ). The IRAK1/4 and JNK INHs, alone or in combination, produced similar inhibitions in AP-1 activity, indicating that they are in the same pathway for IL-1 ␤ production.
A recent report also shows that PKC ␦ mediates high glucose-induced sterile infl ammatory response by upregulating nuclear factor kappa B and infl ammatory cytokine expression in monocytic cells ( 21 ).Our results indicate that both classical PKC (PKC ␣ and ␤ ) and PKC ␦ play an important role in IL-1 ␤ secretion, as both general Ro-31-8220 and classical PKC INH Go6976 signifi cantly attenuated Ox-LDL-induced IL-1 ␤ production. However, because Ro-31-8220, Rottlerin, and PKC ␦ siRNA inhibited Ox-LDLinduced IRAK1 kinase activity, it can be concluded that PKC ␦ is the main PKC mediating IRAK1-induced IL-1 ␤ production. PKC ␦ operates upstream of the IRAK-JNK-AP-1 Our fi ndings have implications for sterile infl ammatory disorders because signifi cant increases in Ox-LDL and IL-1 ␤ were observed in SIRS patients, which positively correlated with each other. Low and high Ox-LDL-containing plasma of healthy and SIRS patients primed monocytes for IL-1 ␤ overproduction by activating PKC ␦ and IRAK1 in a CD36-, TLR2-, TLR4-, and TLR6-dependent manner. PKC ␦ is thus proposed to be an attractive target for preventing IL-1 ␤ production and sterile infl ammation observed during chronic infl ammatory disorders.
Ox-LDL induced time-dependent CD36 upregulation and activation of the IRAK pathway, thus indicating the possible role of CD36 and TLRs in Ox-LDL-induced IL-1 ␤ production.
Therefore, we checked the role of these receptors in Ox-LDL-induced PKC ␦ and IRAK1 activation and IL-1 ␤ production. For studying the effect of Ox-LDL on cell lines expressing higher amounts of CD36, we used PMA-differentiated THP1 cells, which express high amounts of this receptor ( 46 ) and were also used in studies such as ( 7 ). Gene silencing with TLR2-, TLR4-, TLR6-, or CD36-specifi c siRNA in THP1 monocytes and macrophages signifi cantly attenuated Ox-LDL-induced PKC ␦ and IRAK1 activation and IL-1 ␤ production, suggesting that TLR2, TLR4, TLR6, and CD36 can mediate Ox-LDL-induced effects seen in the present study. Interestingly, PKC ␦ positively regulated Ox-LDL-induced CD36 upregulation. Therefore, inhibition in CD36 upregulation due to less PKC ␦ may also contribute to the reduced signaling events transduced by these receptors. However, there are several ways by which TLRs can be activated. Ox-LDL can induce CD36-dependent TLR4 and TLR6 heterodimerization during IL-1 ␤ production, as shown in THP1 monocytic cells ( 6 ). TLR2, TLR4, or TLR6 can also heterodimerize and interact with CD36 in a ligand-specifi c manner ( 54 ). Ox-LDL can also prime monocytes and peripheral blood mononuclear cell for cytokine overproduction by upregulating TLR2 and TLR4 ( 4,55,56 ).
Because a positive correlation existed between circulating Ox-LDL and IL-1 ␤ in both SIRS and healthy subjects, it can be speculated that increased circulating Ox-LDLs can be a factor for enhanced IL-1 ␤ production in humans. A positive correlation between Ox-LDL and IL-1 ␤ with the disease severity scores (APACHE II and SOFA) also indicated an association of Ox-LDL concentration with the extent of IL-1 ␤ production and disease severity.
The effect of low and high Ox-LDL-containing plasma of control and SIRS individuals on PKC ␦ and IRAK1 activation and IL-1 ␤ production was CD36-dependent, because blocking this receptor by CD36 FA6 antibody signifi cantly attenuated these signaling events. Because CD36 FA6 antibody completely blocks binding of Ox-LDL to CD36 receptor ( 57 ), it can be speculated that plasma-induced IL-1 ␤ production due to Ox-LDL will be minimal in the antibody-treated samples. A previous report also suggests that Ox-LDLinduced IL-1 ␤ production is attenuated in monocytes derived from CD36-defi cient patients ( 58 ).
Although experiments done with specifi c siRNA demonstrate the role of TLR2, TLR4, TLR6, and CD36 in SIRS high Ox-LDL plasma-induced PKC ␦ and IRAK1 activation and IL-1 ␤ production, these results can be interpreted in several ways. Plasma can be a source of several entities, including IL-1 ␤ that may induce PKC ␦ and IRAK1 activation and cytokine overproduction. At the same time, some studies suggest a role of minimally modifi ed LDL in IL-1 ␤ production by upregulating and activating TLR2 and TLR4 ( 3,59,60 ), and its presence in the plasma cannot be ruled out .