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The Journal of Immunology, 2007, 179, 8480-8490
Copyright © 2007 by The American Association of Immunologists, Inc.
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Caspase-4 Interacts with TNF Receptor-Associated Factor 6 and Mediates Lipopolysaccharide-Induced NF-{kappa}B-Dependent Production of IL-8 and CC Chemokine Ligand 4 (Macrophage-Inflammatory Protein-1β)1

Umayal Lakshmanan and Alan G. Porter2

Cell Death and Human Diseases, Genomics and Genetics Division, Institute of Molecular and Cell Biology, Proteos, Singapore, Republic of Singapore


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human caspase-4 does not have a corresponding mouse ortholog. Caspase-4 falls within the class of "inflammatory caspases," being homologous with human caspases 1 and 5 and mouse caspases 1, 11, and 12. To address the function of caspase-4, we generated caspase-4-deficient human THP1 monocytic cell lines which exhibited substantially reduced LPS-induced secretion of several chemokines and cytokines, including IL-8 (CXCL8), CCL4 (macrophage-inflammatory protein-1β), CCL20 (macrophage-inflammatory protein-3{alpha}), and IL-1β. The LPS-induced expression of the mRNAs encoding these cytokines was correspondingly reduced in the caspase-4-deficient clones. Because a specific NF-{kappa}B inhibitor blocked LPS-induced IL-8 and CCL4 mRNA expression as well as IL-8 and CCL4 secretion in THP1 cells, we investigated the role of caspase-4 in NF-{kappa}B signaling. LPS-induced NF-{kappa}B nuclear translocation and activation were inhibited in all caspase-4-deficient clones. LPS stimulation led to the interaction of endogenous caspase-4 and TNFR-associated factor 6 (TRAF6) via a TRAF6-binding motif (PPESGE), which we identified in caspase-4. Mutation of this site in caspase-4 resulted in the loss of the TRAF6-caspase-4 interaction. Similar TRAF6-binding motifs are known to be functionally important for TRAF6 interactions with other molecules including caspase-8, and for mediating NF-{kappa}B activation in various immune and nonimmune cell types. Our data suggest that the TRAF6-caspase-4 interaction, triggered by LPS, leads to NF-{kappa}B-dependent transcriptional up-regulation and secretion of important cytokines and chemokines in innate immune signaling in human monocytic cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Caspases, chiefly associated with apoptosis, were discovered more recently to have diverse nonapoptotic roles (1). In mammals, these range from inflammation—caspases 1, 5, 11, 12 (2, 3, 4, 5); cell differentiation—caspases 3, 8, 14 (6, 7, 8, 9, 10, 11, 12, 13, 14); proliferation and suppression of immune cell development—caspases 8 and 3, respectively (15, 16, 17, 18, 19); and NF-{kappa}B activation—caspases 1, 2, 8, 10 (20, 21, 22, 23, 24, 25, 26). Some of the nonapoptotic roles are evolutionary conserved; for example, the Drosophila caspase DREDD has a prominent role in innate immunity (27). Moreover, it is not surprising that one protein may have multiple functions, given the advanced complexity in mammals achieved with proportionally fewer genes compared with lower organisms.

Following its initial cloning (28, 29, 30), caspase-4 was without an assigned function until recently, when it was shown to have a role in endoplasmic reticulum (ER)3 stress-mediated apoptosis (31, 32), though this has been disputed (33). Phylogenetic classification (34) places human caspase-4 in a group along with caspases 1, 5, 11, and 12, found only in vertebrates, and named the inflammatory caspases based on the prototype of this group caspase-1. This group does not have orthologs in the fly and nematode, suggesting that they evolved together with a complex hemopoietic system (34). Caspase-4 is sandwiched between caspase-1 and pseudocaspase-12 on human chromosome 11, whereas caspase-11 is located between caspase-1 and caspase-12 on mouse chromosome 9. Caspases 4 and 5 in humans are believed to have arisen by gene duplication of mouse caspase-11. Moreover, procaspase-4 is 59% identical with procaspase-11, which is involved in cytokine maturation in the mouse (4). Also, the mRNAs of caspases 4 and 11 have similar tissue distribution patterns (35). Thus, a dual role for caspase-4 in apoptosis and inflammation is anticipated.

Inflammation is essentially the effects of rapidly produced cytokines in response to the activation of the innate immune system, the first line of defense against pathogens (36). This evolutionarily ancient innate immune response helps control infections at an early stage by activating proinflammatory signaling cascades and also by playing an important role in triggering and shaping the adaptive immune response through up-regulation of cytokines and costimulatory molecules (37). The innate immune system uses a conserved set of pattern recognition receptors called TLRs to recognize specific sets of pathogen-associated molecular patterns (PAMPs) found in infectious microorganisms (36). PAMPs from each class of microbes are unique and conserved structures that are indispensable for their survival. One such PAMP is LPS, the cell wall component of Gram-negative bacteria, and the receptor involved in its recognition is TLR4. Like the other 10 known human TLRs, TLR4 has a unique extracellular domain for recognizing the LPS signal, together with a highly homologous intracellular Toll/IL-1R (TIR) domain (38). The TIR is responsible for downstream signaling culminating in activation of several transcription factors, notably NF-{kappa}B which regulates the transcription of cytokines and other proinflammatory mediators such as IL-8 (CXCL8) and CCL4 (MIP-1β), thereby acting as a master switch for inflammation (39). TLR4 with its TIR domain interacts with the adaptor molecule MyD88/Mal, which in turn recruits members of the IL-1R-associated kinase (IRAK) family. The serine-threonine kinases, IRAK1 and IRAK4, cross-phosphorylate each other, which results in recruitment of TNFR-associated factor 6 (TRAF6) and its translocation to the cytosol. TRAF6 interacts with UBC13 and UEV1A, and these proteins form a ubiquitin-conjugating enzyme (E2) for which TRAF6 serves as an ubiquitin ligase (E3). TRAF6 either activates the kinase transforming growth factor β-activated kinase-1 (TAK-1), which in turn activates the inhibitor of I{kappa}B kinase (IKK) complex leading to NF-{kappa}B activation, or phosphorylates the kinases MKK3 and MKK6 leading to p38 MAPK and JNK activation (40).

In the absence of a murine homolog of caspase-4, we used human cell lines to address the role of caspase-4 in the innate immune responses to LPS. A particular advantage of using cells from humans compared with mice is the frequent and substantial species differences in their immune systems; for example, the absence of an exact homolog of human IL-8 in mice (41, 42). Using small hairpin RNA (shRNA) knockdown technology, we generated stable human monocytic cell lines with massively reduced expression of caspase-4, and demonstrated that caspase-4 plays an important role in the classical LPS-induced TLR4-signaling pathway that leads to NF-{kappa}B activation and consequent up-regulation and secretion of IL-8 and CCL4.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

The human monocytic cell lines, THP1 (ATCC TIB-202) and U937 (ATCC CRL-1593.2; American Type Culture Collection), were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified incubator with 5% CO2. HEK 293T human embryonic kidney cell line (a highly transfectable derivative of the 293 cell line into which the temperature-sensitive gene for SV40 T-Ag was inserted) was maintained in DMEM medium supplemented with heat-inactivated FBS and penicillin-streptomycin antibiotics. All reagents were obtained from Sigma-Aldrich except for those mentioned below. LPS was obtained from Alexis, hygromycin B was obtained from BD Biosciences, and the NF-{kappa}B inhibitor BMS-345541 was obtained from Calbiochem. Transfections of THP1 cells and 293T cells were conducted using Fugene 6 and Lipofectamine 2000, respectively. The Abs were obtained from various sources: caspase-4 and caspase-8 from MBL International Corporation; TRAF6, IRAK1, actin, c-myc, and NF-{kappa}B p65 from Santa Cruz Biotechnology; lamin B Abs from Calbiochem; phospho-Abs and secondary Abs from Cell Signaling; and anti-FLAG Ab from Sigma-Aldrich. Fugene 6 was obtained from Roche and Lipofectamine 2000 was obtained from Invitrogen Life Technologies.

Stable knockdown of caspase-4

Target sites in caspase-4 for designing shRNAs to knockdown casp-4 were considered only in regions common to all the major isoforms of caspase-4 that lacked any homology to other proteins in the human genome. Using the small interfering RNA target finder tool from the Ambion website, three targets were synthesized, annealed, and ligated into the linearized pSilencer vector (Ambion) containing the hygromycin selection marker. The following two regions were efficient targets in knocking down caspase-4: 1), GGA GCA CCT TCA TTA GTA C and 2) CGA CTG TCC ATG ACA AGA T. The clones used in the following experiment were developed from the stable cells expressing the antisense RNA against the first target. The cloned shRNA target in the vector was transfected into THP1 cells by electroporation using the Gene Pulser (Bio-Rad) together with the negative vector control (VC) containing a scrambled sequence (from Ambion) with no homology to any sequence in the human or mouse genomes. Stable clones selected over several weeks in the presence of hygromycin at 500 µg/ml were screened for caspase-4 knockdown by Western blot. Positive clones were maintained in RPMI 1640 medium containing 50 µg/ml hygromycin.

Human cytokine array, RT-PCR, and ELISA

Rapid analysis of the secreted cytokines and chemokines was performed using the Raybio kit from Raybiotech, and spots were quantified with a Bio-Rad densitometer using the software Quantityone. For RT-PCR analysis, total RNA was isolated by the TRIzol method using the TRI reagent from Molecular Research Center and cDNA was synthesized using the Superscript first-strand synthesis kit from Invitrogen Life Technologies. The SingleGene PCR kit from SuperArray Bioscience was used for gene-specific PCR expression analysis of the following cytokines or chemokines, viz., IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-11, IL-12A, IL-12B, IL-18, IFN-{gamma}, CCL4 (MIP-1β), CCL20 (MIP-3{alpha}), CCL2 (MCP-1), CCR2, M-CSF, CCL5 (RANTES), and TNF-{alpha}, along with GAPDH, the internal control for normalization. Agarose gel images were scanned and analyzed using a Bio-Rad densitometer and Quantityone software. Real-time RT-PCR analysis of cytokine mRNAs was done using the RT2 quantitative PCR primers with the SYBR green quantitative real-time PCR technology (SuperArray Bioscience). The SYBR green PCR master mix (Promega) was used to set up the reaction in a Corbett research thermal cycler with the following program: 95°C, 15 min; 40 cycles of (95°C, 30 s; 55°C, 30 s; and 72°C, 30 s). GAPDH and 18s rRNA were used for normalization. For ELISA quantification of secreted cytokines, the Quantikine kits obtained from R&D Systems and the Biotrak system obtained from Amersham Biosciences were used.

Reporter assays, confocal microscopy, and fractionation

The NF-{kappa}B reporter gene (provided by Dr. L. Li; Institute of Molecular and Cell Biology, Singapore) and the Renilla luciferase reporter (provided by Dr. S. Dhakshinamoorthy; Institute of Molecular and Cell Biology, Singapore) in the pRL-TK plasmid were used in a Dual Luciferase reporter system to quantify NF-{kappa}B activity in the transfected cell lysates using a TD-20/20 luminometer (Promega). Cells grown in chamber slides were fixed with 3.7% paraformaldehyde solution for 20 min at 24°C. Cells were then washed with PBS and blocked with 1% normal goat serum, diluted in PBS containing 0.1% Triton X-100 for 2 h at 24°C. The primary Ab against p65 (1/100 dilution) was added to fresh blocking solution and the cells incubated at 4°C overnight. They were washed three times for 5 min each with PBS containing 0.1% Triton X-100 before incubation with anti-rabbit FITC-labeled secondary Ab (Molecular Probes) at room temperature for 2 h. Cells were again washed with PBS containing 0.1% Triton X-100 before being mounted in Vectashield (Vector Laboratories). Hoechst 33342 (Molecular Probes) was diluted 1/5000 in PBS and was used to stain the nuclei before mounting. Cells were observed and photographed using a Bio-Rad MRC 2000 confocal microscope connected to a Photometrics Sensys CCD camera. Nuclear fractionation to identify the nuclear translocated p65 subunit of NF-{kappa}B was conducted using the nuclei isolation kit from Panomics.

Coimmunoprecipitation

The modified Radioimmunoprecipitation Assay buffer (43) for THP1/U937 cells (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% deoxycholic acid, 0.1% SDS with complete Roche protease inhibitor without EDTA tablet-1 per 25 ml) was used to immunoprecipitate the TRAF-6/caspase-4/IRAK1 complex using the immunoprecipitation protocol from the Sefton laboratory (http://pingu.salk.edu/~sefton/Hyper_protocols/immunoprecip.html). Briefly, cells were harvested in ice-cold tubes, centrifuged, and washed with ice-cold PBS followed by lysis in 500 µl of THP1/U937 immunoprecipitation buffer or 293T immunoprecipitation buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1% Triton X-100 with Roche protease inhibitor tablet without EDTA-1 per 25 ml) for 20 min on ice, and the lysate was cleared at 20,000 x g for 30 min. After saving a portion for total lysate analysis and quantification, the remainder was used for incubation with the respective Ab for 1 h on ice. A total of 50 µl of anti-mouse IgG beads (eBioscience) was added and incubated on a rocking platform at 4°C for 2 h. After three to four washes with 1 ml of the respective lysis buffer, the beads were stored dry at –20°C until further analysis. Freshly prepared sample buffer with DTT was used to solubilize the proteins, and after boiling for 3 min followed by centrifugation, the sample was immediately loaded and analyzed by Western blot. The mouse TrueBlot Western blot kit (eBioscience) protocol was used to mask the H and L chains of the Ab.

Site-directed mutagenesis

Mutants A, B, and C (TRAF6-binding site (TBS) in caspase-4) were created one by one using the QuikChange II XL site-directed mutagenesis kit from Stratagene in the pEGFP plasmid from BD Clontech. The sequences of mutants A, B, and C were, respectively, PPASGE (CCACCTGCGTCAGGAGAA), APASGE (GCACCTGCGTCAGGAGAA), and AASGA (GCACCTGCGTCAGGAGCA). The endotoxin-free plasmid DNA kit obtained from Machery-Nagel was used to prepare the DNA for transfection following purification with the Qiagen plasmid kit.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Stable knockdown of caspase-4 in the human monocytic cell line THP1

Fig. 1A is a schematic showing that the human casp-4 gene encodes three major isoforms, {alpha}, {gamma}, and {delta}, from nine exons spanning a region of ~25 kb. A short hairpin RNA, driven by U6 RNA polymerase, was designed to target all the three major isoforms without affecting any other known proteins. An almost complete knockdown in basal caspase-4 protein levels was observed in four independent stable clones (Fig. 1B). In contrast, the yield of caspase-8 remained essentially the same in all four clones (Fig. 1B), which as discussed later is significant because caspase-8 has been shown to be involved in both innate and adaptive immunity through NF-{kappa}B activation (15, 17, 22).


Figure 1
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  		FIGURE 1. Stable knockdown of caspase-4 in human monocytic THP1 cells. A, Representation of the region of human chromosome 11 encoding the casp-4 transcripts. The three major mRNAs ({alpha}, {gamma}, {delta}) transcribed from this region are shown with their exons depicted as part-shaded or striped boxes interspersed by introns. The shRNA depicted by the black rectangular box (see Materials and Methods for sequence) targets all three mRNAs because it is located in the last exon common to all three. B, Western blot. Top panel, Stable and efficient knockdown of caspase-4 by shRNA in four independent THP1 clones (TA, TB, TC, TD). The parental clones are denoted as WT and the VC clones stably express shRNA against a scrambled sequence. Middle panel, Western blot probed with a caspase-8 Ab.

 
Defects in secreted cytokines in the caspase-4 knockdown clones

Caspase-4 belongs to a group of caspases involved in inflammation, and hence we investigated the role of this caspase in cytokine production. Caspase-4 knockdown and control caspase-4-expressing cells were treated with LPS (a TLR4 inducer) for 8 h and the pattern of 72 secreted cytokines and chemokines was examined using a semiquantitative commercial Ab array. Knockdown of caspase-4 altered the yields of several extracellular cytokines (including some chemokines) after TLR4 induction with LPS—notably, IL-8 and CCL4—highlighted in Fig. 2A, hepatocyte growth factor, epidermal growth factor, CCL5 (RANTES), and CXCL7 (NAP-2) (data not shown). The amounts of IL-8 and CCL4 spots on the array were markedly suppressed (by 1.7-fold for both) in the caspase-4 knockdown clone after LPS treatment (Fig. 2A). The reduction in extracellular IL-8 and CCL4 protein levels in the caspase-4 knockdown clones was confirmed and accurately quantified using an ELISA, which showed a 5- and 6-fold reduction in immunoreactive IL-8 and CCL4, respectively (Fig. 2B). A similar substantial reduction in extracellular CCL20 (MIP-3{alpha}) was observed in the caspase-4 knockdown clones (data not shown). Together, these results suggest the possibility that caspase-4 has a role upstream of secretion of certain inflammatory cytokines and chemokines induced by LPS.


Figure 2
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  		FIGURE 2. Defects in secreted cytokines in the caspase-4 knockdown clones. A, THP1 parental (WT, left panel) and caspase-4 knockdown shRNA clone cells (TC, right panel) were treated with LPS for 8 h, and the supernatants were subjected to the "Raybio" protein array to analyze the secretion pattern of 72 cytokines. The IL-8 and CCL4 spots, highlighted in octagons, were scanned by densitometry, and the intensities of the spots were compared before and after LPS treatment. B, ELISA to quantify and compare IL-8 and CCL4 in the culture supernatants of THP1 clones. Caspase-4 knockdown clones (TA, TB, TC, TD) along with THP1 WT and VC cells were treated with LPS for 8 h, and the supernatants were used to quantify the IL-8 levels (left panel) and CCL4 levels (right panel). Data are presented as "control" comprising WT or VC, and "clones" comprising the four caspase-4 shRNA clones, and represented as mean ± SE. Values of p were <0.001 for both sets of clones in B compared with WT and VC.

 
Reduced LPS-stimulated up-regulation of specific cytokine mRNAs in caspase-4 knockdown cells

Cytokines are tightly regulated at three major steps (44, 45): transcription, processing, and secretion. Caspase-1 plays an essential role in the processing of cytokines like IL-1β, IL-18, and IL-33 (46, 47, 48). We investigated the step at which caspase-4 might participate in regulating cytokine production by using semiquantitative RT-PCR as an initial screen to determine whether the knockdown in caspase-4 had any effect on LPS-induced mRNA yields of a wide spectrum of cytokines and chemokines. Fig. 3, A and B, shows that following LPS stimulation for 4 h, 6 of the 15 tested mRNAs (IL-1β, IL-6, IL-8, IL-12B, CCL4, and CCL20) all exhibited a consistent 50% or greater reduction after LPS stimulation in four independent caspase-4-deficient clones compared with the parental and VC cells. The remaining mRNAs encoding IL-18, IL-2, IL-4, IL-5, IL-10, IFN-{gamma}, and CCL2 showed no consistent reduction, so were not investigated further (Fig. 3, A and B). We chose IL-8 and CCL4 for most further studies, because their mRNAs and secreted proteins were present in substantially lower yields in the caspase-4 knockdown clones as judged by both the RT-PCR quantification (Fig. 3B), Ab array (Fig. 2A), and ELISA (Fig. 2B). To confirm the RT-PCR results and to verify that the decreased yields of the mRNAs for IL-8 and CCL4 are due to a reduction in LPS-induced mRNA up-regulation, we repeated the RT-PCR analysis both before and after LPS treatment of THP-1 cells and their caspase-4 knockdown counterparts (Fig. 4, A and C). Whereas there was no difference in the basal, untreated levels of IL-8 and CCL4 mRNAs, there were consistent ~50% reductions in both mRNAs following 4 h of LPS stimulation in all the caspase-4-deficient clones (Fig. 4, A and C). A similar reduction in the mRNA for CCL20 was observed in all the caspase-4 knockdown clones following LPS stimulation (data not shown). To further verify these findings, we conducted quantitative real-time RT-PCR, which clearly showed that IL-8 and CCL4 mRNAs were poorly up-regulated by LPS in the caspase-4-deficient cells compared with the wild-type (WT) and VC cells (Fig. 4, B and D). Although TNF mRNA was only marginally reduced in the caspase-4 knockdown clones in the semiquantitative RT-PCR, quantitative PCR did confirm that TNF mRNA production is dependent in large part on caspase-4 (data not shown). Together with the other data in Figs. 3 and 4, these results indicate lower LPS-induced yields of some cytokine and chemokine mRNAs in a caspase-4-deficient human monocytic cell line, and demonstrate that the reduced LPS induction of IL-8 and CCL4 mRNAs mirrors the deficiency of their secreted proteins under similar conditions (Fig. 2B; and CCL20, data not shown).


Figure 3
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  		FIGURE 3. Reduced LPS-stimulated yields of specific cytokine mRNAs in caspase-4 knockdown cells. A, Each panel shows the LPS-stimulated mRNA level of a different cytokine mRNA as judged by RT-PCR and separated by agarose gel electrophoresis. From left to right: parental (WT); TA, TB, TC, TD caspase-4 knockdown, and VC THP1 cells after LPS treatment for 4 h. The panels marked with an asterisk (*) on the left side indicate the cytokine mRNAs that are down-regulated in the caspase-4 knockdown clones. B, Quantification by densitometry of selected mRNAs that appear to be down-regulated in A (*). {blacksquare}, Cytokine mRNA levels in WT and VC; {square}, cytokine mRNA levels in the caspase-4 knockdown clones. Data in B are represented as mean ± SE of the control or clones after grouping their respective densitometric volumes together. Values of p were: IL-1β, p = 0.02; IL-6, p = 0.07; IL-8, p = 0.04; IL-12B, p = 0.02; CCL4, p = 0.01; CCL20, p = 0.04.

 

Figure 4
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  		FIGURE 4. Reduced LPS-stimulated up-regulation of specific cytokine mRNAs in caspase-4 knockdown cells. A, Top panel, RT-PCR was used to detect IL-8 mRNA yields before (Untr) and after LPS treatment for 4 h. Lower panel, Quantification of RT-PCR shown in left panel by densitometry after normalization against GAPDH. Controls: mean values of parental and VC cells. shRNA clones: mean values of caspase-4 knockdown clones, TA to TD. The p values before and after LPS treatment were 0.66 and 0.0003, respectively. B, Real-time RT-PCR quantification of IL-8 mRNA in WT, VC cells, and the shRNA clones TB and TC, before and after 4 h of LPS stimulation. Values are mean ± SE of two individual samples. Values of p were ≤0.05 for all the clones treated with LPS compared with both WT + LPS as well as VC + LPS. C, Corresponding data for the LPS-induced up-regulation of CCL4 mRNA derived as in A. Because the internal control GAPDH primers are not compatible with the CCL4 primers, the GAPDH primer data from A was used for normalization. Values are the mean ± SE of the controls grouped together comprising WT and VC, and the caspase-4 shRNA group comprising the clones TA to TD. The p values before and after LPS treatment were 0.64 and 0.01, respectively. D, Real-time RT-PCR to quantify CCL4 mRNA before and after LPS stimulation of the same cells as in B. Values of p were ≤0.001 and ≤0.0005 for caspase-4 shRNA clones treated with LPS compared with WT + LPS and VC + LPS, respectively.

 
Caspase-4 mediates LPS-induced IL-1β production

We initially observed reduction in the levels of IL-1β mRNA in the caspase-4 knockdown clones after LPS stimulation (Fig. 3A and B). This was confirmed by semiquantitative RT-PCR after LPS treatment of the parental, VC, and four caspase-4 shRNA clones (Fig. 5A). To further verify these findings, we conducted real-time RT-PCR, which clearly showed that IL-1β mRNA was only slightly up-regulated by LPS in the caspase-4-deficient cells compared with the WT and VC cells (Fig. 5B). IL-1β requires two distinct signals for its activation and release (44, 49). The first signal is triggered by LPS and the second signal can be derived from the activation of cell surface purinergic receptors of the P2X7 subtype, for which ATP is the main endogenous ligand (49, 50). Hence, after LPS stimulation for 6 and 8 h, we treated the parental and caspase-4 knockdown cells either with or without 7 µM ATP for 30 min before harvesting the supernatant for ELISA quantification of secreted IL-1β (Fig. 5C). The results confirm that ATP is required for IL-1β secretion, and indicate that caspase-4 deficiency compromises the synthesis and hence the ATP-dependent secretion of IL-1β (Fig. 5C).


Figure 5
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  		FIGURE 5. Synthesis of IL-1β mRNA and secretion of IL-1β are compromised in caspase-4 knockdown clones. A, Left panel, RT-PCR to determine IL-1β mRNA in untreated and LPS-treated (4 h) samples of caspase-4 knockdown clones TA, TB, TC, and TD along with the controls (WT, VC). Right panel, Quantification of levels of up-regulated IL-1β mRNA after normalizing for GAPDH levels and averaging the controls (WT, VC) and caspase-4 knockdown clones. B, Real-time RT-PCR quantification of IL-1β mRNA in WT, VC cells, and the shRNA clones TB and TC, before and after 4 h of LPS stimulation. Values of p were ≤0.05 and ≤0.01 for shRNA clones treated with LPS compared with WT + LPS and VC + LPS, respectively. C, Secreted IL-1β levels as estimated by ELISA in the supernatants of caspase-4 knockdown clones (TB, TC) and the controls (WT, VC) at 6 and 8 h after LPS treatment (1 µg/ml) in the absence or presence of 7 µM ATP for 30 min. Values of p for both time points were ≤0.002.

 
Caspase-4 mediates LPS-induced cytokine induction through NF-{kappa}B-dependent transactivation

LPS signaling through TLR4 converges mainly on NF-{kappa}B transcription factor activation and subsequent cytokine transcription (51, 52). Having provided evidence that caspase-4 is involved in the transcriptional up-regulation of certain cytokines and their mRNAs through the TLR4 pathway, we examined whether caspase-4 is required for NF-{kappa}B activation (Fig. 6). All four caspase-4 knockdown clones had markedly suppressed LPS-mediated induction of NF-{kappa}B activity as indicated by the >50% reduced reporter activity after LPS treatment (Fig. 6B). This suppression was corroborated by confocal microscopy, which showed nuclear translocation of the NF-{kappa}B p65 subunit was selectively compromised in the four caspase-4 knockdown clones at various times following LPS stimulation (Fig. 6A1, quantified in Fig. 6A2). The impaired nuclear translocation of the NF-{kappa}B p65 subunit was further confirmed by cell fractionation experiments, which demonstrated that LPS-induced p65 protein translocation was strongly impaired in all the caspase-4 knockdown clones compared with the caspase-4-expressing parental and VCs (Fig. 7A, quantification in lower panel of 7A).


Figure 6
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  		FIGURE 6. Reduced NF-{kappa}B activation and nuclear translocation of the p65 subunit of NF-{kappa}B in caspase-4 knockdown cells. A1, Translocation of p65 NF-{kappa}B subunit to the nucleus was delayed and reduced in caspase-4 knockdown clones TB and TC compared with WT and TVC THP1 cells as shown by confocal imaging of the NF-{kappa}B p65 subunit at different times after LPS stimulation. p65 is stained green with FITC-conjugated secondary Ab. The nucleus is stained blue with Hoechst 3334. The top two panels of TB are shown at a lower magnification A2 quantification of means of at least six independent fields from two treatments for each time point. Statistical analysis using the two-tailed t test comparing the parental and VC against the two knockdown clones gave the following p values: 0.4 before treatment; 0.09 at 5 min; 0.0008 at 15 min; and 2.5E-05 at 45 min after LPS treatment. B, NF-{kappa}B transcriptional activity determined by luciferase reporter activity 4 h after LPS stimulation of THP1 (WT) and two VC cell lines (TVC4, TVC5), along with the four independent caspase-4 shRNA clones (TA to TD). Mean and SE of triplicates of each treatment and transfection are represented as the fold induction after 4 h compared with the zero time after normalization with Renilla for the differential transfection efficiencies. Values are the mean ± SE of three independent transfections. Two-tailed t test analysis of the caspase-4 shRNA clones against the three controls gave a p value <0.0001.

 

Figure 7
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  		FIGURE 7. Further evidence of reduced NF-{kappa}B activation in caspase-4 knockdown cells. A, Upper panel, Western blot comparing cytosolic and nuclear levels of NF-{kappa}B p65 subunit after LPS treatment for the various indicated times in control (WT, VC) and caspase-4 knockdown clones (TA to TD). The blot was reprobed with lamin B to control for the purity of the nuclear fractions. Lower panel, Densitometric quantification of the upper middle panel, in which the WT and VC nuclear p65 values (controls) were combined, and the nuclear p65 values (clones) were also combined. The bars represent the mean ± SE. Two-tailed t test analysis of the caspase-4 shRNA clones and controls (WT + VC) gave p values of 0.02 before treatment; 0.01 at 15 min; 0.003 at 30 min; and 0.26 at 60 min of LPS treatment. B, WT (left panel) and caspase-4 shRNA clones (right panel) were treated with LPS for the various indicated times, and the yield of I{kappa}B{alpha} was examined by Western blot with an I{kappa}B{alpha} phosphospecific Ab (P-I{kappa}B{alpha}, top panels) or an I{kappa}B{alpha}-specific Ab (I{kappa}B{alpha}, second row panels). Third row panels, The LPS-induced yields of phospho-IKKβ (P-IKKβ). Fourth and fifth row panels, IKK{alpha} and IKKβ, which are involved in NF-{kappa}B signaling, serve as loading controls.

 
NF-{kappa}B p65 subunit translocation is usually preceded by the phosphorylation and degradation in the cytoplasm of I{kappa}B{alpha}, the inhibitor of nuclear translocation of the RelA (p65) subunit (53). Whereas the LPS-induced phosphorylation of I{kappa}B{alpha} occurred strongly and at early times after LPS stimulation of caspase-4-expressing cells, there was inhibition of and delay in the phosphorylation and resultant degradation of I{kappa}B{alpha} protein in a caspase-4 knockdown clone (Fig. 7B, top two panels). I{kappa}B{alpha} phosphorylation requires the kinase activity of IKK{alpha}/IKKβ, which in turn needs to be phosphorylated for activation. Consistent with the pattern exhibited above, IKKβ phosphorylation was almost completely absent in the caspase-4 knockdown clones unlike in the control cells (Fig. 7B, third row panels). As loading controls, IKK{alpha} and IKKβ protein levels remained unchanged over time (Fig. 7B, lowest two panels).

It is established that NF-{kappa}B-dependent transcription of a number of cytokine mRNAs contributes importantly to the subsequent production of cytokines including IL-8 (39, 51, 52). Therefore, to verify the role of NF-{kappa}B activation in IL-8 (and CCL4) signaling after LPS stimulation of THP1 cells, we treated caspase-4-expressing THP1 cells with a NF-{kappa}B inhibitor BMS-345541, which potently inhibited the LPS-induced transcription of mRNAs encoding IL-8 and CCL4 (Fig. 8A) and the secretion of immunoreactive IL-8 and CCL4 proteins (Fig. 8, B and C, respectively). As a control, U0126 a specific inhibitor of the MEK1/2 pathway, failed to suppress LPS-induced IL-8 and CCL4 mRNA up-regulation or IL-8 and CCL4 protein production (Fig. 8, A–C). Altogether, these data are evidence that caspase-4 acts upstream of and is required for NF-{kappa}B activation that in turn leads to intracellular IL-8/CCL4 mRNA up-regulation and IL-8/CCL4 protein production in the cell culture medium.


Figure 8
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  		FIGURE 8. NF-{kappa}B inhibitor blocks LPS-induced up-regulation and secretion of IL-8 and CCL4 (A) RT-PCR analysis of IL-8 and CCL4 mRNAs after LPS stimulation for 4 h in THP1 parental cells treated with a vehicle control (DMSO), or an NF-{kappa}B pathway inhibitor (I{kappa}K inhibitor BMS345541), or MEK1/2 inhibitor (U0126). Two-tailed statistical analysis comparing the effect of DMSO and the two inhibitors after LPS treatment gave the following values: p = 0.002 for BMS345541 and p = 0.07 for U0126 in the case of IL-8, and p = 0.002 for BMS345541 and p = 0.98 for U0126 in the case of CCL4. B and C, ELISA quantification of IL-8 and CCL4 levels, respectively, in the THP1 culture supernatant after 4 h of LPS stimulation using identical treatments as in A.

 
Caspase-4 interacts with endogenous and transfected TRAF6

LPS triggers TLR4 resulting in recruitment of the adapters MyD88 or MyD88-adapter-like to the activated receptor, which in turn promotes the association of IRAK1 and TRAF6 leading to activation of a NF-{kappa}B-signaling complex (39, 51, 52). Coimmunoprecipitation experiments were performed with caspase-4, TRAF6, and IRAK1 Abs to determine whether caspase-4 might be a part of this multiprotein signaling complex in THP1 and U937 monocytic cells. Following LPS stimulation, the interaction of endogenous caspase-4 with endogenous TRAF6 in THP1 cells was an extremely early event, peaking at 5–10 min after LPS stimulation before returning to uninduced levels at 60 min (Fig. 9A, upper panel). Furthermore, there was a reproducible second peak of interaction starting at ~2 h after LPS stimulation in THP1 cells. With U937 cells, there was also a clear and strong interaction of caspase-4 and TRAF6 at 5 min after LPS stimulation (Fig. 9B, upper panel). As has been established previously (54), TRAF6 and IRAK1 were found to interact in THP1 and U937 cells following the expected kinetics of association from 5 to 20 min after LPS treatment (Fig. 9, A and B, second panels). Endogenous caspase-4 also interacted with IRAK1 in both cell lines, closely following the biphasic pattern of the TRAF6-caspase-4 interaction in THP1 cells (Fig. 9, A and B, fourth panels). However, the kinetics of the IRAK1-caspase-4 interaction was slightly delayed compared with the caspase-4-TRAF6 association (Fig. 9, A and B), suggesting that the TRAF6-caspase-4 interaction occurs just before the association of IRAK1 with caspase-4 in the signaling complex.


Figure 9
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  		FIGURE 9. LPS induces endogenous caspase-4 to interact transiently with endogenous TRAF6 and IRAK1. A, THP1 cells after treatment with LPS for various times were subjected to immunoprecipitation with TRAF6 or IRAK1 Abs, and immunoblotted with caspase-4 or TRAF6 or IRAK1 Abs as indicated on the Western blots. B, U937 monocytic cells after treatment with LPS for various times were subjected to immunoprecipitation with TRAF6 or IRAK1 Abs, and immunoblotted with caspase-4 or TRAF6 or IRAK1 Abs using the Trueblot kit from eBioscience (Materials and Methods).

 
The cellular interaction of caspase-4 and TRAF6 was further confirmed in a different system using tagged proteins transfected in an alternative cell type 293T. Transfected FLAG-TRAF6 was able to pull down endogenous as well as transfected myc-tagged caspase-4 in a coimmunoprecipitation experiment (Fig. 10, lane 3), whereas in contrast, myc-tagged caspase-1 failed to interact with TRAF6 (Fig. 10, lane 5).


Figure 10
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  		FIGURE 10. TBS in caspase-4 is essential for LPS-induced TRAF6-caspase4 interaction. 293T cells were transfected with TRAF6-FLAG, caspase-4-c-myc, active site mutant caspase-4-c-myc, control caspase-1-c-myc or TBS mutants A, B, or C (Table I) in various combinations as shown in the key at top. Thirty-six hours after transfection, cells were treated with LPS for 10 min and subjected to immunoprecipitation with FLAG Ab and immunoblotted with caspase-4 or c-myc Ab. Bottom two panels, Input levels of transfected TRAF6-FLAG and caspase-c-myc proteins.

 
Intact TRAF6-binding motif in caspase-4 important for TRAF6-caspase-4 association

Examination of caspase-4 reveals the presence of a putative TRAF6-binding motif (PPESGE) at amino acids 95–100, which probably lies in a surface location in the protein according to the Swiss model program (55, 56). Table I, TRAF6-binding motif section, shows that PPESGE is similar to other TRAF6-binding sequences (57), having an absolutely conserved proline and glutamic acid in the P–2 and P0 positions, as well as a well-conserved glutamic acid in the P3 position. To examine the possibility that caspase-4 participates in TLR4 signaling by acting as a binding partner of TRAF6, we generated one to three mutations in the TBS of caspase-4 (A–C series, Table I, Caspase-4-TBS mutants section) and assessed their ability to coimmunoprecipitate TRAF6. Consistent with the interactions of endogenous caspase-4 and TRAF6 (Fig. 9), FLAG-TRAF6 interacted with both c-myc-tagged parental caspase-4 (Fig. 10, lane 3) and active site mutant caspase-4 (Fig. 10, lane 6). In contrast, caspase-4 mutants with single, double, or triple mutations in the conserved residues of the putative TRAF6-binding motif failed to coimmunoprecipitate with FLAG-TRAF6 (Fig. 10, lanes 7–9). As further evidence of the specificity of the TBS for TRAF6, we showed that the enzymatically inactive mutant of caspase-4 (Cys to Ser in the active site) was still able to bind TRAF6 efficiently (Fig. 10, lane 6).


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  		Table I. Comparison of TBS motifs in TRAF6-interacting partners along with TBS of caspase-h and its mutantsa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
LPS, the cell wall component of infecting Gram-negative bacteria, stimulates TLR4 to signal through different transcription factors (notably NF-{kappa}B) to transcribe cytokines and chemokines like IL-8, IL-1β, CCL4, and CCL20. These and other secreted proteins play important roles in innate immunity by eliciting inflammation and triggering the adaptive arm of immunity (58). Human IL-8, the prototypical human chemokine, has critical roles in acute inflammation, motogenesis, angiogenesis, tumorigenesis, and metastasis (42, 59). During inflammation, CCL4, a member of the CC chemokine family, exhibits potent chemoattractant activity for T lymphocytes, NK cells, and immature dendritic cells; moreover, CCL4 can block HIV-1 infection as both CCL4 and HIV-1 share the same receptor, CCR5 (60).

By substantially knocking down caspase-4 expression with shRNAs, we showed that caspase-4 is required to produce a significant proportion of IL-8, CCL4, IL-1β, and CCL20 in the culture supernatant of a human monocytic cell line following LPS stimulation. It is established (61, 62) and we confirmed that LPS-induced production of IL-8 and CCL4 proceeds mainly via NF-{kappa}B-dependent transcription of their genes. IL-1β is another important cytokine that is activated by two distinct signals (44, 49), and the mechanisms that regulate its synthesis, maturation, and secretion are of great interest in the context of various inflammatory diseases. As a component of the inflammasome, caspase-1 has an indispensable role in IL-1β maturation (3); here, we report the involvement of caspase-4 as an additional protease with a key role in the production of IL-1β. It is noteworthy that while caspase-1 is required for the cleavage and maturation of pro-IL-1β, caspase-4 acts much more upstream of caspase-1 by mediating the NF-{kappa}B-dependent transcription of the gene encoding IL-1β.

Our evidence suggests that the production of IL-8 and CCL4 depends to a significant extent on caspase-4 acting upstream of LPS-induced, NF-{kappa}B-dependent transcriptional activation of their mRNAs (see model in Fig. 11). At what point and by which mechanism could caspase-4 function between TLR4 and NF-{kappa}B? Ligand stimulation of TLR4 attracts the adaptor protein MyD88, which recruits and facilitates the phosphorylation and consequent activation of the receptor-associated kinase IRAK1. This leads to poly ubiquitination by a TRAF6:E3 ligase complex in the cytosol (36, 40, 63), which stimulates the activity of the associated TAK1 kinase (64, 65). The activated TAK1 complex phosphorylates the kinase IKKβ, which in turn phosphorylates I{kappa}B to facilitate its degradation resulting in activation of NF-{kappa}B required for transactivation of various cytokines (52). Importantly, TRAF6 is required for LPS signaling in vivo (66). We showed that endogenous caspase-4 (but not caspase-1) interacts with TRAF6 through a well-conserved TRAF6-binding motif that is known to be functionally important for TRAF6 interactions with other molecules, and for mediating NF-{kappa}B activation in various immune and nonimmune cell types (67, 68). Our findings together with these considerations strongly suggest that the TRAF6-caspase-4 interaction may be necessary for LPS-induced NF-{kappa}B activation in human monocytic cells.


Figure 11
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  		FIGURE 11. Speculative model showing the position occupied by caspase-4 in LPS signaling. The LPS-induced interaction of caspase-4 and TRAF6 in a complex with IRAK1 stimulates the signaling pathway leading to I{kappa}B{alpha} degradation and NF-{kappa}B nuclear translocation that is required for the transcription and production of IL-8 and CCL4. The dotted arrow between the caspase-4/TRAF6/IRAK1 complex and IKKβ indicates that the mechanisms by which caspase-4 contributes to NF-{kappa}B activation are not known.

 
IL-1β, IL-8, CCL4, and CCL20 mRNAs are poorly up-regulated by LPS in caspase-4-deficient cells, yet we did not go so far as to examine the LPS-induced levels of all cytokine mRNAs that are known to be regulated by LPS. Thus, we cannot exclude the existence of caspase-4-independent pathways to the production of certain cytokines and chemokines. Indeed, there is an additional TRAF6-independent pathway in TLR4 signaling which requires the recruitment of TIR domain containing adapter inducing IFN-β (TRIF) and TRIF-related molecule adaptor proteins and subsequent induction of NF-{kappa}B, IFN regulatory factor-3, or AP-1 transcription factors (51, 69). It is established that TLR4 stimulation results not only in the activation of NF-{kappa}B, but also the stimulation of many additional pathways including the protein kinases PI3K, p38, JNK, MEKK3, TPL2, ASK1, ERK, and TBK1, which lead to induction of AP-1, IRF3, or IRF5 factor-dependent transcription (38, 51, 69). The picture is complex, and it is unclear to what extent gene expression mediated by these other kinases and transcription factors requires (or is independent of) NF-{kappa}B. Nevertheless, the TLR4-mediated production of at least some cytokines, chemokines, and IFNs does involve transcription factors other than NF-{kappa}B in monocytic cells and macrophages.

It is interesting that caspases 3, 5, 7, 8, 10, 11, and 12 each have at least one potential TBS (our unpublished observations). This raises the question of whether any or all of these caspases are involved in some aspects of immune cell regulation through their interactions with TRAF6, a central mediator of signaling through several different TLRs (66). In fact, caspase-8 is involved in mediating T cell proliferation via TCR signaling, which is dependent on caspase-8 interacting with TRAF6 through a TBS (43, 70). Human caspase-10 which is closely related to caspase-8 in homology and function (71) might therefore also use a TBS interaction for its signaling. Caspase-3 has been shown to have several roles in immune cell development (18, 19), and this could also potentially involve TRAF6 signaling. Caspases 5 and 11, with known roles in the inflammasome complex and LPS-induced cytokine up-regulation, respectively (3, 4), could be anticipated to have such a site for interactions with TRAF6 and subsequent signaling events. Only full-length human caspase-12, which is enzymatically inactive, has the TBS while the short-truncated form present in the vast majority of the human population lacks this site. It has been shown that full-length inactive caspase-12 dampens endotoxin responsiveness through the production of fewer cytokines (5, 72). This begs the question of whether the enzymatically inactive full-length human caspase-12 might compete with caspase-4 for TRAF6 binding, thereby suppressing LPS-induced caspase-4-dependent cytokine regulation and endotoxin responsiveness in a dominant-negative fashion.

Caspase-8 has a role in B cell-mediated immunity and TCR-dependent T cell proliferation (15, 22, 73) and signals via its interaction with TRAF6 (43, 70). However, we believe it is unlikely that caspase-8 is also involved together with caspase-4 in LPS-induced NF-{kappa}B activation and cytokine production in THP1 monocytic cells. This is because we showed that procaspase-8 protein was synthesized normally in the caspase-4 knockdown clones, which displayed defective NF-{kappa}B activation and cytokine production. Caspase-1 is expressed in THP-1 cells, but caspase-1 lacks a TBS, consistent with our observation that it fails to interact with TRAF6 (2, 4). In caspase-1 knockout mice, pro-IL-1β mRNA is synthesized normally in the cytosol; however, pro-IL-1β processing and secretion fail to occur (2). These considerations together suggest that while caspase-1 plays a role mainly in the LPS-induced processing of cytokines as a component of the inflammasome protein complex, caspase-4 is essential for the synthesis of IL-1β.

A picture has emerged of nonapoptotic caspase function in which only a small fraction of the caspase pool is recruited to signaling complexes, which can involve caspase clustering with (or without) cleavage and removal of the prodomain—a step normally required for full caspase proteolytic activation (26, 74). Enzymatically active caspase-8 is essential in TCR-mediated NF-{kappa}B activation (17, 43), whereas in contrast the enzymatic activity of caspase-8 is dispensable for TNF-induced NF-{kappa}B activation (22). Using NF-{kappa}B reporter and ELISA, we were not able to address the questions of 1) whether enzymatically active caspase-4, or 2) the TRAF6-caspase-4 interaction, are required for LPS-induced NF-{kappa}B activation and cytokine secretion. This is because transfection of procaspase-4 did not stimulate further LPS-induced NF-{kappa}B activation and cytokine secretion compared with endogenous procaspase-4, strongly suggesting that the levels of endogenous procaspase-4 in THP1 cells are already saturating (our unpublished observations). In support, only a small fraction of the endogenous procaspase-4 pool is likely to be recruited to TRAF6 in a signaling complex. There are many other open questions. What are the partners and substrates of caspases 4 and 8 in immune cell signaling? Does caspase-4 participate in stimulating ubiquitination of TRAF6 that leads to NF-{kappa}B activation, perhaps by acting on some component(s) of the TRAF6-signaling complex, such as TAK1, TAB1, TAB2, UEV1A, or UBC13?

Addressing the issue of which is the closest human ortholog of murine caspase-11, our study reinforces the idea that both caspase-4 and caspase-5 have caspase-11 as their ancestor (34, 75). Both caspases 4 and 5 share some characteristics of caspase-11, like inducibility after LPS treatment in the case of caspase-5 (76) and participation in innate immune responses in the case of both caspases 4 and 5 (our results and Ref. 75). Yet caspase-4 also resembles caspase-12 in its ability to contribute to apoptosis induced by ER-mediated stress (31, 32), though this remains controversial (33). It seems, therefore, that human caspase-4 and caspase-5 have acquired additional diverse functions during their evolution. Thus, caspase-5 is involved in sensing endogenous danger signals via the inflammasome complex, for example, leading to caspase-1 proteolytic processing and IL-1β and IL-18 production (3), whereas caspase-4 participates in a classical receptor-dependent innate immune response to LPS and possibly ER stress responses. The fact that caspase-4 regulates human IL-8, which also lacks an obvious homolog in mice (42), further reinforces this view of the relatively recent evolution of this class of caspases in mammals.

Finally, caspase-8 deficiency leads to various immune-related diseases in different animal and human disease models (16); therefore, it would be interesting to examine other immune-deficient diseases in humans to determine whether caspase-4 is down-regulated or mutated.


   Acknowledgments
 
We thank Vinay Tergaonkar for helpful advice and for reviewing the manuscript. A. G. Porter is an adjunct staff member of the Department of Surgery, National University of Singapore.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funds from A*STAR to the Institute of Molecular and Cell Biology, Singapore. Back

2 Address correspondence and reprint requests to Dr. Alan G. Porter, Cell Death and Human Diseases, Genomics and Genetics Division, Institute of Molecular and Cell Biology, Proteos, 61, Biopolis Drive, Singapore 138673, Republic of Singapore. E-mail address: mcbagp{at}imcb.a-star.edu.sg Back

3 Abbreviations used in this paper: ER, endoplasmic reticulum; PAMP, pathogen-associated molecular pattern; TIR, Toll/IL-1R; IRAK, IL-1R-associated protein; TAK, transforming growth factor β-activated kinase-1; TRAF6, TNFR-associated factor 6; TBS, TRAF6-binding site; VC, vector control; WT, wild type; IKK, I{kappa}B kinase; shRNA, small hairpin RNA. Back

Received for publication July 16, 2007. Accepted for publication October 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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