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Pseudomonas aeruginosa Delays Kupffer Cell Death via Stabilization of the X-Chromosome-Linked Inhibitor of Apoptosis Protein This information is current as of October 1, 2021. Alix Ashare, Martha M. Monick, Amanda B. Nymon, John M. Morrison, Matthew Noble, Linda S. Powers, Timur O. Yarovinsky, Timothy L. Yahr and Gary W. Hunninghake J Immunol 2007; 179:505-513; ;

doi: 10.4049/jimmunol.179.1.505 Downloaded from http://www.jimmunol.org/content/179/1/505

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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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Pseudomonas aeruginosa Delays Kupffer Cell Death via Stabilization of the X-Chromosome-Linked Inhibitor of Apoptosis Protein1

Alix Ashare,2* Martha M. Monick,* Amanda B. Nymon,* John M. Morrison,* Matthew Noble,* Linda S. Powers,* Timur O. Yarovinsky,* Timothy L. Yahr,† and Gary W. Hunninghake*‡

Kupffer cells are important for bacterial clearance and cytokine production during infection. We have previously shown that severe infection with Pseudomonas aeruginosa ultimately results in loss of Kupffer cells and hepatic bacterial clearance. This was associated with prolonged hepatic inﬂammation. However, there is a period of time during which there is both preserved hepatic bacterial clearance and increased circulating TNF-␣. We hypothesized that early during infection, Kupffer cells are protected against TNF-␣-induced cell death via activation of survival pathways. KC13-2 cells (a clonal Kupffer cell line) were treated with P. aeruginosa (strain PA103), TNF-␣, or both. At early time points, TNF-␣ induced caspase-mediated cell death, Downloaded from but PA103 did not. When we combined the two exposures, PA103 protected KC13-2 cells from TNF-␣-induced cell death. PA103, in the setting of TNF exposure, stabilized the X-chromosome-linked inhibitor of apoptosis protein (XIAP). Stabili- zation of XIAP can occur via PI3K and Akt. We found that PA103 activated Akt and that pretreatment with the PI3K inhibitor, LY294002, prevented PA103-induced protection against TNF-␣-induced cell death. The effects of LY294002 included decreased levels of XIAP and increased amounts of cleaved caspase-3. Overexpression of Akt mimicked the effects of PA103 by protecting cells from TNF-␣-induced cell death and XIAP cleavage. Transfection with a stable, nondegradable http://www.jimmunol.org/ XIAP mutant also protected cells against TNF-␣-induced cell death. These studies demonstrate that P. aeruginosa delays TNF-␣-induced Kupffer cell death via stabilization of XIAP. The Journal of Immunology, 2007, 179: 505–513.

he hepatic reticuloendothelial system (RES)3 plays an im- actively phagocytose and kill bacteria but do not produce a large portant role in removing bacteria and endotoxin from the inflammatory response. T blood stream. Kupffer cells, the resident macrophages of We have previously shown that hepatic bacterial clearance by the hepatic RES, provide a crucial means of defense against mi- Kupffer cells is decreased during severe bacteremia with Pseudo- croorganisms. Although they were once thought to be uniform, monas aeruginosa (3). This loss of hepatic bacterial clearance by guest on October 1, 2021 Kupffer cells are now known to be a heterogeneous group of mac- could be exacerbated by Kupffer cell ablation with gadolinium rophages. They are 2-fold more abundant in the periportal region, chloride and prevented by pretreatment with the nonspecific where blood enters the liver (1). Periportal Kupffer cells are larger caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluorometh- and have greater phagocytic function but appear to be less in- ylketone. A notable difference between mild bacteremia (where volved in inflammatory reactions. The smaller centrilobular hepatic bacterial clearance was preserved) and severe bacteremia Kupffer cells appear to be more important in generating inflam- was that severe bacteremia was associated with a prolonged in- matory responses. Recently, a clonal cell line of murine periportal crease in hepatic TNF-␣, suggesting that hepatic inflammation Kupffer cells (KC13-2 cells) has been generated (2). These cells may be involved in the eventual loss of bacterial clearance. Inter- estingly, there was a period of time during severe P. aeruginosa bacteremia during which hepatic bacterial clearance persisted *Division of Pulmonary, Critical Care, and Occupational Medicine, and †Department despite significant inflammation. This lead to the hypothesis of of Microbiology, University of Iowa Roy J. and Lucille A. Carver College of Med- these studies that early in infection, P. aeruginosa protects icine and ‡Veterans Administration Medical Center, Iowa City, IA 52242 Kupffer cells from TNF-␣-induced cell death via activation of Received for publication February 5, 2007. Accepted for publication April 14, 2007. survival pathways. The costs of publication of this article were defrayed in part by the payment of page P. aeruginosa has previously been shown to prevent apoptosis charges. This article must therefore be hereby marked advertisement in accordance in corneal epithelial cells by activation of the epidermal growth with 18 U.S.C. Section 1734 solely to indicate this fact. factor (EGF) receptor (4). More recently, Pseudomonas syringae, 1 This work was supported by a Veterans Affairs Merit Review Grant, by National a plant pathogen, has been shown to prevent apoptosis by trans- Institutes of Health Grants HL-60316, HL-077431, and HL079901-01A1 (to G.W.H.); K12 RR017700 and K08 DK073519-01A1 (to A.A.); and RR00059 from location of an ubiquitin ligase protein (5). Our data demonstrate the General Clinical Research Centers Program, National Center for Research Re- that P. aeruginosa promotes KC13-2 cell survival in the setting of sources, National Institutes of Health. inflammation via stabilization of the X-chromosome-linked inhib- 2 Address correspondence and reprint requests to Dr. Alix Ashare, Division of Pul- monary, Critical Care and Occupational Medicine, University of Iowa, 200 Hawkins itor of apoptosis protein (XIAP). Drive, C33GH, Iowa City, IA 52242. E-mail address: [email protected] The inhibitor of apoptosis protein (IAP) are a family of eight 3 Abbreviations used in this paper: RES, reticuloendothelial system; EGF, epidermal proteins that block the activity of caspases-3, -7, and -9 (6–8). growth factor; IAP, inhibitor of apoptosis protein; XIAP, X-chromosome-linked IAP; XIAP is the most potent IAP, blocking caspase activity with nano- Z-DEVD-FMK, Z-D(OMe)-E(OMe)-V-D(OMe)-FMK; T3S, type III secretions. molar affinity (7). TNF-␣ induces cleavage of XIAP to an inactive Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 fragment (9). Recently, XIAP was found to be phosphorylated by www.jimmunol.org 506 KUPFFER CELLS AND XIAP

FIGURE 1. PA103 protects Kupffer cells from early TNF-␣-induced cell death. A, ATP viability assay was performed after incubation of KC13-2 cells with PA103, TNF-␣, or both (left). PA103 was protective against TNF-␣- ␣-p Ͻ 0.05, TNF ,ء .induced cell death alone compared with TNF-␣ with PA103. Values reflect mean Ϯ SE of three separate experiments. Staining KC13-2 cells with ethidium homodimers after incubation with PA103, TNF-␣, or both confirms the protective effect of PA103 (right). The number of cells positive for ethidium per 100 cells counted is expressed as percent of cell death. B, ATP viability assay comparing PA103 ExsA⍀, TNF-␣, or both shows protection by PA103 ExsA⍀. p Ͻ 0.05, TNF-␣ alone compared Downloaded from ,ء with TNF-␣ with PA103 ExsA⍀. Values reflect mean Ϯ SE of three separate experiments. C, ATP viability assay comparing LPS (600 ng/ml), TNF-␣, or both shows no protection against TNF-␣-induced cell death by http://www.jimmunol.org/ LPS (left). Values reflect mean Ϯ SE of three separate experiments. ATP viability assay comparing TNF-␣, smooth PAO, rough PAO, or smooth or rough PAO in combination with TNF-␣ shows improved survival in cells treated with rough PAO and TNF-␣ or smooth PAO and TNF-␣ p Ͻ ,ء .compared with TNF-␣ alone

0.05) (right). Values reﬂect mean Ϯ by guest on October 1, 2021 SE of three separate experiments.

Akt, a kinase downstream of PI3K, rendering it resistant to formed according to the manufacturer’s instructions. The caspase-3 inhib- cleavage (10). We demonstrate that inhibition of PI3K blocked itor, Z-D(OMe)-E(OMe)-V-D(OMe)-FMK (Z-DEVD-FMK) was obtained the protective effect of P. aeruginosa on KC13-2 cell death. from R&D Systems. A stock solution of 2 mM concentration in DMSO was diluted in PBS before use in cell culture. The final concentration in cell Conversely, overexpression of Akt protected cells against TNF- culture was 20 ␮M. ␣-induced cell death. Finally, transfection with a mutant XIAP that is resistant to cleavage resulted in protection of KC13-2 cells against TNF-␣. These data illustrate that P. aeruginosa Cell culture protects Kupffer cells against caspase-mediated cell death early KC13-2 cells were a gift from Professor R. Landman (University Hospital, via stabilization of XIAP. Furthermore, there may be a window Basel, Basel, Switzerland; see Ref. 2). These cells were cultured as pre- of opportunity in P. aeruginosa infection during which Kupffer viously described in conditioned RPMI 1640 medium containing 1% Glu- cells are protected, preventing the eventual loss of hepatic bac- tamax, 1% nonessential amino acids, 1% sodium pyruvate, 0.15% HEPES, terial clearance. 5% heat-inactivated FCS, and 10% volume of supernatants from the human hepatocyte cell line HepG2 and the human endothelial cell line EAhy926 (2). P. aeruginosa strains PA103, PA103 ExsA⍀, PAO smooth (AK857), Materials and Methods and PAO rough (AK1012) were provided by Dr. T. Yahr (Department of Reagents Microbiology, University of Iowa Roy J. and Lucille A. Carver College of Chemicals were obtained from Sigma-Aldrich. The PI3K inhibitor Medicine, Iowa City, IA). For studies involving live strains of P. aerugi- LY294002 was obtained from Calbiochem. LY294002 was resuspended in nosa, bacteria were cultured overnight at 37°C in Luria-Bertani broth. Bac- ethanol and used at a final concentration of 20 ␮M. Abs were from various teria were subcultured the following morning in Luria-Bertani broth, grown sources: anti-XIAP Abs were from AbCam and Cell-Signaling; Abs to to log phase, quantified based on OD600, and confirmed by standard plat- cleaved caspase-3 and total and phosphorylated Akt were also from Cell ing. The multiplicity of infection in all experiments was 0.5. For experi- Signaling; the Ab to ␤-actin was from Sigma-Aldrich. Secondary Abs for ments using cell-free supernatants of P. aeruginosa, bacteria were cultured Western analysis were from Santa Cruz Biotechnology. Recombinant mu- overnight in KC13-2 medium at 37°C and subcultured in KC13-2 medium rine TNF-␣ was obtained from R&D Systems and had Ͻ1.0 endotoxin U the following morning. Bacteria were grown to the late stationary phase per ␮g of cytokine. TNF-␣ was dissolved in PBS and used at a final con- and centrifuged at low speed; culture supernatants were sterile filtered. The centration of 40 ng/ml. Escherichia coli LPS was obtained from List Bi- final supernatant was added to the cell culture in 25% of the total volume. ological Laboratories. E. coli LPS was used at a final concentration of 600 For experiments using heat-killed supernatants or LPS, the supernatants or ng/ml. ELISA kit for IL-6 was obtained from R&D Systems and was per- LPS were boiled at 100°C before use. The Journal of Immunology 507

FIGURE 2. PA103 stabilizes XIAP and prevents TNF-␣-induced Kupffer cell death at early time points. A, Western blot for XIAP shows that PA103 prevents the TNF-␣-induced reduction in XIAP protein levels at 90 min. ␤-Actin is included as a loading control. Values representative of three experiments and densitometry comparing all three Western blots show that TNF-␣ results in a signiﬁcant reduction ␣-p Ͻ 0.05, TNF ,ء .in XIAP protein compared with PA103 and TNF-␣. B, Western blots demonstrate the time course of Akt activation following PA103, TNF-␣, or both. These Western blots demonstrate that PA103 prolongs the activation of Akt following TNF-␣.

Values are representative of three ex- Downloaded from periments. C, Western blot for cleaved caspase-3 shows that PA103 prevents the TNF-␣-induced increase in cleaved caspase-3 at 90 min (left). Active caspase-3 assay via flow cytometry using a fluorescent substrate confirms that PA103 protects against TNF-␣-induced http://www.jimmunol.org/ caspase-3 activation at 90 min (right). p Ͻ 0.05, TNF-␣ alone compared ,ء with TNF-␣ with PA103.

Cell survival assays Quantitative real-time PCR with primers specific for P. aeruginosa was by guest on October 1, 2021 performed as previously described (3). Dead cells were visualized by adding ethidium homodimer (Invitrogen Life Technologies) to cells in culture to a final dilution of 1/250 and evaluated Adenovirus vectors using a fluorescent inverted microscope after 5 min. A total of 500 cells were counted in 5 fields of view. Cellular ATP was measured using the First-generation recombinant adenovirus vectors were generated by the CellTiter-Glo Luminescent Viability Assay (Promega) and was performed University of Iowa Gene Transfer Vector Core (11) with the exception of according to the manufacturer’s instructions. the myristylated constitutively active Akt vector (Ad-myr-Akt), which was provided by Dr. K. Walsh (Boston University, Boston, MA). The construc- Cleaved caspase-3 assay tion (12) and activity (12, 13) of the Ad-myr-Akt vector have been described previously. The particle titers of the adenoviral stocks were typi- Caspase-3 activity was measured using the CaspGLOW Red Active cally 1012 DNA particles/ml; functional titers were ϳ2 ϫ 1010 PFU/ml. Caspase-3 Staining kit (BioVision Research Products) and was performed Adenovirus vectors expressing the transgene for GFP (AdGFP) driven by according to the manufacturer’s instructions using Guava PCA flow cy- the cytomegalovirus promoter, Ad-myr-Akt, or an empty vector containing tometer (Guava Technologies). no transgene (Ad-EV), were used to infect the cells at a multiplicity of infection of 100. These vectors were free of wild-type virus contamination Western blot analysis by both plaque assay and PCR. KC13-2 cells were plated, and virus was Western blot analysis for specific proteins was performed on total cellular added in serum-free medium. The cells were incubated at 37°C for 2 h, and protein isolated from KC13-2 cells. Protein concentrations in lysates were then medium containing serum was added. The cells were incubated over- measured using the Bradford assay. Twenty micrograms of protein were night and stimulated the following morning according to each individual mixed 1:1 with 2ϫ sample buffer (20% glycerol, 4% SDS, 10% 2-ME, experimental protocol. Either cells were evaluated for viability or protein 0.05% bromphenol blue, and 1.25 M Tris, pH 6.8) and separated using was harvested. Efficiency of infection was determined in each experiment SDS-PAGE. Cell proteins were transferred to polyvinylidene difluoride by examining green fluorescence of the AdGFP-infected cells using an Ͼ membranes (Bio-Rad). Equal loading of proteins was evaluated using Pon- inverted fluorescent microscope and was 75% in all experiments. Similar ceau S dye staining (Sigma-Aldrich). The polyvinylidene difluoride mem- infection efficiencies were assumed with the other adenovirus vectors. brane was saturated with methanol, washed, and then incubated with pri- Plasmid transfections mary Ab overnight at 4°C. Blots were washed four times and incubated with HRP-conjugated anti-IgG Ab (1/5,000–1/40,000). Immunoreactive XIAP-S87D was a generous gift from Dr. Jin Cheng (University of South bands were developed using a chemiluminescent substrate, ECL, and ECL Florida, Tampa, FL) (10). Plasmids were transfected using FuGENE 6 Plus (Amersham Biosciences) and detected by autoradiography. Protein reagent (Roche Molecular Systems) according to the manufacturer’s in- levels were quantified using densitometry via a FluorS scanner and Quan- structions with a 3:2 ratio of FuGENE 6 reagent to DNA. tity One software for analysis (Bio-Rad). Densitometry is expressed as mean and SE of three separate Western blots. Flow cytometry Quantitative real-time PCR KC13-2 cells were grown to 70% confluency and cotransfected with a GFP plasmid and either XIAP-S87D or an empty plasmid. 48 h after transfec- Cells were treated as described. Bacterial DNA was isolated using the Bugs tion, cells were stimulated as described in the specific experimental pro- NЈ Beads kit (Genpoint) according to the manufacturer’s instructions. tocol. At set time points, cells were harvested, fixed, permeabilized, and 508 KUPFFER CELLS AND XIAP stained with PE-labeled Ab against cleaved caspase-3 (Cell Technology). Cytometric analysis looking at GFP- and PE-stained cells was performed using an LSR flow cytometer (BD Biosciences). Statistical analysis Statistical analyses were performed on cellular ATP data using ANOVA followed by Bonferroni’s test for multiple comparisons and represent the mean Ϯ SEM. These methods were performed using GraphPad Prism (Prism). Results P. aeruginosa protects Kupffer cells against early cell death To evaluate the relative effects of P. aeruginosa and TNF-␣ on Kupffer cell survival, KC13-2 cells were treated with P. aeruginosa (strain PA103), TNF-␣, or both. This strain is a known human pathogen and produces epithelial cell injury via the elaboration of type III secreted toxins (14). We used a final concentration of 40 ng/ml TNF-␣ in all experiments. This is the peak concentration of serum TNF-␣ in a murine model of severe P. aeruginosa sepsis (3). Because ATP amounts have been shown to correlate with metabolically active (viable) cells (15), we measured cellular Downloaded from ATP as a marker of viability. We found that at 90 min, PA103 protected KC13-2 cells against TNF-␣-induced cell death (Fig. 1A, left). This was manifested as preservation of cellular ATP levels. Use of ethidium homodimers to assess plasma membrane perme- ability confirmed the early effect of PA103 on KC13-2 cell survival (Fig. 1A, right). TNF-␣ was dissolved in PBS and studies FIGURE 3. Inhibition of caspase-3 improves Kupffer cell survival and http://www.jimmunol.org/ enhances bacterial clearance. A, KC13-2 cells were treated with TNF-␣, the looking at PBS alone showed no effect on KC13-2 cell survival caspase-3 inhibitor Z-DEVD-FMK 20 ␮M, or both (with Z-DEVD-FMK (data not shown). pretreatment initiated 30 min before incubation with TNF-␣). ATP viabil- PA103 has been shown to induce macrophage apoptosis via the ity assay was performed 6 h after treatment. Treatment with the caspase-3 type III secretion (T3S) system (16). To examine whether the T3S inhibitor with TNF-␣ decreased the amount of ATP loss compared with p Ͻ 0.01). Values reflect mean Ϯ SE of ,ء .system was playing a role in the prosurvival effect of PA103 in this cells treated with TNF-␣ alone model, we infected cells with a mutant strain of PA103 (PA103 three separate experiments. B, KC13-2 cells were pretreated with either 20 ExsA⍀) that is devoid of T3S capabilities (17). We found that the ␮M Z-DEVD-FMK or no inhibitor 30 min before incubation with PA103, PA103 ExsA⍀ strain had the same protective effect of KC13-2 TNF-␣, or both. After 6 h, cells and supernatants were harvested, and by guest on October 1, 2021 cells as the wild-type strain (Fig. 1B). This suggests that the T3S bacterial DNA was isolated. Quantitative real-time PCR with primers system is not required for this effect. specific for P. aeruginosa was performed to determine residual bacterial load. Pretreatment with Z-DEVD-FMK before PA013 and TNF-␣ de- To evaluate whether this was an effect of LPS, we performed the creased the bacterial burden compared with cells treated with PA103 .(p Ͻ 0.05 ,ء) ␣-same experiment using E. coli LPS in place of PA103. We found and TNF that E. coli LPS did not protect KC13-2 cells against TNF-␣-induced cell death (Fig. 1C, left). To ensure that E. coli LPS was functional and able to activate KC13-2 cells, we measured the live replicating bacteria are not required for the protective effect amount of IL-6 in the cell supernatant after incubation with E. coli of P. aeruginosa on KC13-2 cells. To further evaluate whether LPS. We found that E. coli LPS caused an increase in secreted the secreted product responsible for the protective effect of IL-6 as measured by ELISA (data not shown), indicating that E. PA103 was LPS, we boiled cell-free supernatants at 100°C to coli LPS is able to activate KC13-2 cells and induce cytokine denature protein. It is widely accepted that LPS is heat resistant production. However, E. coli LPS does not protect Kupffer cells (19, 20). In fact, a recent study showed that concentrations of from TNF-␣-induced death. LPS as low as 10 ng/ml were effective after boiling for 15 min Because E. coli LPS may not exert the same effect as P. aerugi- (21). We found that PA103 cell-free supernatants that were heat nosa LPS on KC13-2 cells, we treated cells with rough and smooth inactivated had no protection against TNF-␣-induced cell death variants of P. aeruginosa strain PAO. The smooth strain PAO is a (data not shown). As a whole, these data suggest that P. aerugi- common laboratory stain of P. aeruginosa. Similar to PA103, we nosa protects KC13-2 cells from early TNF-␣-induced cell found that PAO protects KC13-2 cells from TNF-␣-induced cell death via a secreted product that is neither LPS nor a member death at 90 min (Fig. 1C, right). The rough variant of PAO lacks of the T3S family. the O Ag and has a poorly functional LPS (18). We found that the ␣ rough variant of PAO also protects KC13-2 cells from TNF-␣- PA103 prevents early TNF- -induced KC13-2 cell death via induced cell death. These data suggest that the protective effect of increased Akt and stabilization of XIAP P. aeruginosa on TNF-induced cell death is not unique to the Because TNF-␣ signaling can result in cleavage of XIAP (9), we PA103 strain of P. aeruginosa and is likely not related to the examined XIAP protein using Western blot. Using an XIAP Ab secretion of LPS. designed to evaluate for the presence of the inactive cleavage prod- To evaluate whether the protective effect of PA103 was me- uct, we found that as early as 1 h after treatment with TNF-␣, there diated by a secreted product, we incubated KC13-2 cells with a was evidence of cleavage of XIAP to its inactive 30kD fragment cell-free supernatant of PA103, TNF-␣, or both. We found that (data not shown). This XIAP cleavage was prevented by PA103. the cell-free supernatant was able to protect KC13-2 cells from We next examined the effect of TNF-␣ and PA103 on XIAP pro- TNF-␣-induced cell death (data not shown). This suggests that tein at time points just before and just after 1 h. We found that at The Journal of Immunology 509

FIGURE 4. Inhibition of PI3K blocks PA103 protection against TNF- ␣-induced Kupffer cell apoptosis. A, ATP viability assay with and without the PI3 kinase inhibitor LY294002 (20 ␮M) shows that the PI3 kinase inhibitor negates the protective effect of PA103 p Ͻ 0.05, LY ϩ PA103 ϩ ,ء .at 90 min TNF-␣ compared with PA103 ϩ TNF-␣. Values reﬂect mean Ϯ SE of three separate experiments. Ethanol control had no effect (data not shown). B, Western blot for phospho-Akt conﬁrms effect of the inhibitor. Western blot for XIAP illustrates that LY294002 prevents the stabilization of XIAP by PA103 in the setting of TNF-␣. Western blot for cleaved caspase-3 shows inhibition

of PI3 kinase prevents PA103-in- Downloaded from duced protection against apoptosis. Values are representative of three experiments. C, Active caspase-3 assay via flow cytometry with a fluorescent substrate confirms the results demonstrated in Fig. 3C.

/p Ͻ 0.05, LY ϩ PA103 ϩ TNF-␣ http://www.jimmunol.org ,ء compared with PA103 ϩ TNF-␣. Values reﬂect mean Ϯ SE of three separate experiments.

30 min, XIAP levels were stable in all groups (data not shown). data indicate that PA103 delays Kupffer cell death following However, at 90 min, there was a decrease in XIAP in the cells TNF-␣ via increased Akt and stabilization of XIAP. by guest on October 1, 2021 treated with TNF-␣. This was prevented by infection with PA103 (Fig. 2A). Inhibition of caspase-3 increases Kupffer cell survival and XIAP is known to be phosphorylated by Akt at Ser87 (10). The enhances clearance of bacteria phosphorylated state of XIAP renders it resistant to TNF-␣-in- We have previously shown that Kupffer cell survival is important duced cleavage. To evaluate whether PA103-induced stabilization for bacterial clearance and that the loss of hepatic bacterial is as- of XIAP was due to increased Akt, we measured phospho-Akt in sociated with increase caspase-3 activity in vivo (3). To test cell lysates following a time course of incubation with PA103, whether inhibition of caspase-3 would protect Kupffer cells in TNF-␣, or both (Fig. 2B). After infection with PA103, Akt activity vitro, we pretreated KC13-2 cells with the caspase-3 inhibitor Z- increases at 30 min and remains activated as long as 2 h. In con- DVED-FMK before incubation with TNF-␣ (Fig. 3A). After6hof trast, TNF-␣ exposure results in an early activation of Akt that incubation with TNF-␣, there was a 40% reduction in cellular ATP starts to decrease by 60 min. Treatment of KC13-2 cells with both compared with control cells, suggesting significant cell death. In TNF-␣ and PA103 results in a more prolonged increase in acti- contrast, pretreatment with Z-DEVD-FMK significantly improved vated Akt compared with TNF-␣ alone. These data are consistent survival of KC13-2 after TNF-␣. DMSO alone had no effect on with PA103 stabilizing XIAP via activation of Akt. Kupffer cell survival (data not shown). To evaluate whether XIAP cleavage was associated with To determine whether improved Kupffer cell survival was as- caspase activation, we measured levels of cleaved caspase-3 pro- sociated with enhanced clearance of bacteria, we treated cells with tein. We found that cleaved caspase-3 was present in the cells either no inhibitor or Z-DEVD-FMK before treatment with PA103, treated with TNF-␣ for 90 min (Fig. 2C, left). This was prevented TNF-␣, or both. Cells were incubated for 6 h, and then bacterial by infection with PA103. To confirm that the cleaved caspase-3 load was measured using quantitative real-time PCR with primers protein levels represent activity, we measured caspase-3 activity specific for P. aeruginosa. We found that treatment with Z-DEVD- via flow cytometry using a fluorescent substrate. We found that FMK before PA103 and TNF-␣ resulted in a decreased bacterial TNF-␣ alone resulted in increased cleaved caspase-3 activity, load of P. aeruginosa compared with cells treated with PA103 and whereas infection with PA103 prevented this effect (Fig. 2C, TNF-␣ alone (Fig. 3B). This is consistent with our prior work right). showing that survival of Kupffer cells is critical for maintenance of Because our prior study demonstrated eventual Kupffer cell bacterial clearance. death after PA103 bacteremia, we evaluated whether XIAP stabilization by PA103 was transient. We found that at later time points, Inhibition of PI3 kinase prevents PA103-induced protection of PA103 was no longer protective against TNF-␣-induced XIAP Kupffer cells cleavage (data not shown). This corresponded with lack of protec- We next evaluated whether inhibition of PI3 kinase with the tion against TNF-␣-induced cell death (data not shown). These inhibitor LY294002 was sufficient to prevent PA103-induced 510 KUPFFER CELLS AND XIAP

Overexpression of Akt prevents TNF-␣-induced Kupffer cell apoptosis To examine the effect of active Akt overexpression on TNF-␣- induced Kupffer cell death, we infected cells with an adenovirus expressing a myristylated form of Akt which is constitutively active. We found that cells treated with Ad-myr-Akt and TNF-␣ had improved survival compared with cells treated with TNF-␣ alone or empty vector with TNF-␣ (Fig. 5A). To evaluate the efﬁcacy of the viral vector, we measured protein levels of total and active Akt. We found that treatment with Ad-myr-Akt increased levels of total and active Akt in KC13-2 cells with or without TNF-␣ (Fig. 5B) compared with control cells or cells treated with the empty vector. We next examined the effect of Ad-myr-Akt on XIAP and cleaved caspase-3 in KC13-2 cells. We found that Ad-myr-Akt stabilized XIAP protein in KC13-2 cells and that this effect was not overcome by treatment with TNF-␣ (Fig. 5C). Furthermore, Ad-myr-Akt prevented caspase-3 activation compared with cells treated with TNF-␣ alone or empty vec- ␣ tor and TNF- . Downloaded from

Overexpression of an XIAP mutant decreases Kupffer cell apoptosis Because Akt expression induces several known survival pathways (22), we examined whether XIAP was necessary to pre-

vent apoptosis in KC13-2 cells. XIAP can be phosphorylated by http://www.jimmunol.org/ Akt at Ser87 to generate a phosphorylated state resistant to cleavage and degradation (10). To determine whether increased XIAP was sufficient to prevent TNF-␣-induced cell death associated with caspase activation in KC13-2 cells, we used a mutant XIAP with an aspartic acid substituted at Ser87 (S87D- XIAP). This mutant mimics the phosphorylated state and is resistant to cleavage. We cotransfected KC13-2 cells with GFP and S87D-XIAP or an empty plasmid followed by treatment with TNF-␣. We evaluated caspase activation by staining cells by guest on October 1, 2021 ␣ FIGURE 5. Overexpression of Akt prevents TNF- -induced Kupffer cell with a PE-labeled cleaved caspase-3 and analyzing cells with apoptosis. A, KC13-2 cells were infected with Ad-EV, Ad-myr-Akt, or no flow cytometry (Fig. 6). We found that transfection with the vector. After 24 h, cells were treated with or without TNF-␣ for 90 min. ATP viability assay shows that pretreatment with the Ad-myr-Akt prevented TNF- empty plasmid resulted in 17.1% of cells cleaved caspase-3 p Ͻ 0.05 compared with Ad-EV ϩ TNF and con- positive, whereas transfection with S87D-XIAP resulted in only ,ء .induced cell death-␣ trol ϩ TNF. Values reflect mean Ϯ SE of three separate experiments. B, 4.7% of cells cleaved caspase-3 positive. These data demon- Western blot for total and phospho-Akt demonstrates that the Ad-myr-Akt strate that stabilization of XIAP increased Kupffer cell survival vector increases levels of Akt protein with or without the presence of TNF-␣. after TNF-␣ exposure. Values are representative of three experiments. C, Western blots for XIAP and cleaved caspase-3 demonstrate that infection with Ad-myr-Akt results in stabilization of XIAP and prevents activation of caspase-3. Values are represen- Discussion tative of three experiments. The hepatic RES is critical for both bacterial clearance and cytokine production in response to systemic infection (23, 24). In severe bacteremia, endotoxin and other bacterial products protection of Kupffer cells. We found that pretreatment with activate subpopulations of Kupffer cells, resulting in liberation LY294002 completely ablated the protective effect of PA103 on of TNF-␣ and other inflammatory mediators (23). These inflam- TNF-␣-induced cell death in KC13-2 cells (Fig. 4A). To examine matory cytokines, whereas necessary to help clear infection, whether this effect was associated with a decrease in XIAP, we promote tissue injury and likely contribute to hepatic apoptosis evaluated levels of XIAP protein in whole cell lysates using and necrosis. We and others have shown that hepatic RES func- Western blot. We found that pretreatment with LY294002 re- tion is impaired during severe bacteremia (3, 25). Here we show sulted in an expected loss of Akt activity and a loss of XIAP that, unlike many other populations of macrophages, periportal protein (Fig. 4B). Kupffer cells are susceptible to TNF-␣-induced cell death. Fur- Subsequently, we examined activated caspase-3 as a marker thermore, we report the novel finding that infection with P. of apoptosis in this model. We found that pretreatment with aeruginosa delays TNF-␣-induced Kupffer cell death by stabiliz- LY294002 resulted in increased cleaved caspase-3 in the cells ing XIAP. treated with TNF-␣ and PA103 compared with those cells Kupffer cells represent the largest population of tissue macro- treated with either PA103 or TNF-␣ alone (Fig. 4B). To confirm phages. Similar to alveolar macrophages, Kupffer cells are derived this, we evaluated caspase-3 activity using a PE-labeled sub- from peripheral blood monocytes that migrate to the liver and dif- strate and found increased caspase-3 activity in cells treated ferentiate into at least two different Kupffer cell populations (1). with LY294002 along with PA103 and TNF-␣ compared with Prior studies have shown that both peripheral blood monocytes and cells treated with PA103 and TNF-␣ alone (Fig. 4C). alveolar macrophages are resistant to TNF-␣-induced apoptosis The Journal of Immunology 511 Downloaded from

FIGURE 6. Stabilization of XIAP protects Kupffer cells against TNF-␣-induced apoptosis. KC13-2 cells were cotransfected with GFP and either an empty plasmid or S87D-XIAP. After 48 h, cells were treated with or without TNF-␣. After 90 min, cells were harvested, ﬁxed, permeabilized, http://www.jimmunol.org/ and stained with PE-labeled Ab against cleaved caspase-3. Flow cytometry was performed to analyze the percentage of cells that were both GFP and PE positive. The panel to the left demonstrates equivalent transfection rates in both groups. This panel also shows that, without TNF-␣, very few cells are PE-positive. In contrast, the panel to the right demonstrates, in the presence of TNF-␣, the cells transfected with the empty plasmid have 17.1% positive for cleaved caspase-3, whereas the cells transfected with S87D-XIAP have only 4.7% positive for cleaved caspase-3. Values are representative of three experiments.

(26, 27). In fact, TNF-␣ has been shown to prevent monocyte Kupffer cells involves a heat-labile secreted product. Potential can- apoptosis in vitro (26). Our data indicate that the large periportal didates would include both phospholipase and exotoxin A. How- by guest on October 1, 2021 Kupffer cells are susceptible to TNF-␣-induced cell death. To our ever, further studies are needed to determine the specific factor(s) knowledge, this is the first report of TNF-␣-induced Kupffer cell responsible for this effect. death in the literature. Taken together with our prior studying XIAP is a potent inhibitor of apoptosis. It directly binds to showing loss of hepatic bacterial clearance in the setting of pro- caspases-3, -7, and -9, rendering them inactive (7, 34, 35). IAPs longed TNF-␣ elevation (3), these data suggest a potential role of are characterized by the presence of one or more baculovirus IAP TNF-␣ in the development of impaired bacterial clearance in repeat domains and a caspase activation recruitment domain. It is sepsis. generally accepted that XIAP is the most potent inhibitor of apo- P. aeruginosa is a virulent human pathogen associated with a ptosis protein (7). In fact, a recent study suggests that two other high mortality. The strain PA103 is particularly virulent due to the members of the IAP family, cIAP1 and cIAP2, bind to caspases presence of a T3S system (28). The T3S system is cytotoxic to but fail to inhibit their activity (36). XIAP, however, binds and epithelial cells (14). Prior studies have implicated P. aeruginosa in inhibits caspases-3, -7, and -9 with nanomolar affinity. Although the induction of macrophage apoptosis. The T3S systems of both the antiapoptotic properties of XIAP have been extensively P. aeruginosa and Yersinia spp. have been shown to mediate mac- studied in malignancy (37–39), very few studies exist evaluat- rophage apoptosis (16, 29). A variety of P. aeruginosa secreted ing the role of XIAP in sepsis. One study demonstrated that products have been shown to induce macrophage apoptosis in cul- increased XIAP was associated with delayed neutrophil apo- ture (30–33). However, these studies did not examine the effect of ptosis in septic patients (40). However, to our knowledge, ours P. aeruginosa on macrophages as early as 1 h after infection. To is the first study to look at the role of XIAP in macrophage our knowledge, this is the first report of PA103 exerting a protec- survival during bacterial infection. tive effect on macrophages. We found that prevention of KC13-2 cell apoptosis by PA103 P. aeruginosa, strain PA01, has been shown to prevent human was mediated by prevention of XIAP cleavage. TNF-␣ and its corneal epithelial cell apoptosis via EGF receptor activation (4). counterpart, TRAIL, are known to induce cleavage and degrada- This is consistent with our observation, given that the EGF recep- tion of XIAP although the exact mechanism remains unclear (9, tor is upstream of PI3K and Akt. The plant pathogen, P syringae, 41, 42). XIAP is regulated by several proteins. In response to mi- prevents apoptosis in tomato plants via the elaboration of a protein tochondrial injury, smac/Diablo and Omi/HtrA2 are released into that mimics the host E3 ubiquitin ligase (5). Our results illustrate the cytosol where they bind and inactivate XIAP (8, 43). This that early protection of Kupffer cells by P. aeruginosa is not results in polyubiquitination and degradation of XIAP. Recently, unique to strain PA103. Furthermore, a key finding in this study is XIAP was found to have an Akt phosphorylation site (10). Phos- that P. aeruginosa protects Kupffer cells early by a mechanism that phorylation at Ser87 stabilizes XIAP, protecting it from degrada- is not mediated by the T3S system or the ability to secrete a fully tion. Here we have shown that phosphorylation of XIAP is suffi- functional LPS. The mechanism of P. aeruginosa protection of cient to promote survival of Kupffer cells treated with TNF-␣. 512 KUPFFER CELLS AND XIAP

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