Activation of TLR4 Is Required for the Synergistic Induction of and Dual Oxidase A2 by IFN-γ and Lipopolysaccharide in Human Pancreatic This information is current as Cancer Cell Lines of September 26, 2021. Yongzhong Wu, Jiamo Lu, Smitha Antony, Agnes Juhasz, Han Liu, Guojian Jiang, Jennifer L. Meitzler, Melinda Hollingshead, Diana C. Haines, Donna Butcher, Krishnendu

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 7, 2013, doi:10.4049/jimmunol.1201725 The Journal of Immunology

Activation of TLR4 Is Required for the Synergistic Induction of Dual Oxidase 2 and Dual Oxidase A2 by IFN-g and Lipopolysaccharide in Human Pancreatic Cancer Cell Lines

Yongzhong Wu,* Jiamo Lu,* Smitha Antony,* Agnes Juhasz,* Han Liu,† Guojian Jiang,* Jennifer L. Meitzler,* Melinda Hollingshead,† Diana C. Haines,‡ Donna Butcher,‡ Krishnendu Roy,† and James H. Doroshow*,†

Pancreatitis is associated with release of proinflammatory cytokines and reactive species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase (Duox)2, an NADPH oxidase essential for –related, gastrointestinal host defense, is regulated by IFN-g–mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because LPS enhances the development and invasiveness of pancreatic cancer in vivo following TLR4- Downloaded from related activation of NF-kB, we examined whether LPS, alone or combined with IFN-g, regulated Duox2. We found that upregulation of TLR4 by IFN-g in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-kB, accumulation of NF-kB (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with small interfering RNAs, as well as two independent NF-kB inhibitors, attenuated LPS- and IFN-g–mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-g and LPS may result from IFN-g–related activation of Stat1 acting in http://www.jimmunol.org/ concert with NF-kB–related upregulation of Duox2. Sustained extracellular accumulation of H2O2 generated by exposure to both LPS and IFN-g was responsible for an ∼50% decrease in BxPC-3 cell proliferation associated with a G1 cell cycle block, apoptosis, and DNA damage. We also demonstrated upregulation of Duox expression in vivo in pancreatic cancer xenografts and in patients with chronic pancreatitis. These results suggest that inflammatory cytokines can interact to produce a Duox-dependent pro- oxidant milieu that could increase the pathologic potential of pancreatic inflammation and pancreatic cancer cells. The Journal of Immunology, 2013, 190: 000–000.

substantial body of evidence suggests that chronic in- inflammation-related tissue injury and reduce subsequent tumor- flammation of the pancreas plays an important role in the igenesis (5). Pancreatic cancer cells were demonstrated to produce by guest on September 26, 2021 A subsequent development of pancreatic cancer and that reactive oxygen species (ROS) in a growth factor–dependent the pathogenesis of exocrine cancers of the pancreas may be inti- fashion, and these reactive species play an important role in the mately related to the release of proinflammatory cytokines and proliferative capacity of these cells (6–8). Therefore, it is possible cytokine-related reactive oxygen formation (1–4). Recently, the that, during repeated bouts of pancreatitis, cytokine-related ROS role of repetitive bouts of asymptomatic pancreatic inflammation in production could increase genetic instability (9, 10) while decreas- tumor development has been emphasized, as well as the critical ing the tumor-suppressor functions of essential phosphatases role of anti-inflammatory interventions to enhance the repair of (11), thus enhancing the possibility of malignant transformation. Although it has been known for more than two decades that tumor cells can produce a significant flux of H2O2 (12), only more *Laboratory of Molecular Pharmacology of the Center for Cancer Research, National recently has it become clear that much of the reactive oxygen Cancer Institute, National Institutes of Health, Bethesda, MD 20892; †Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of formation emanating from human tumors may originate from Health, Bethesda, MD 20892; and ‡Pathology/Histotechnology Laboratory, Science members of the recently described family of epithelial NADPH Applications International Corp. Frederick, Inc./Frederick National Laboratory for oxidases (Noxs): reduced NAD phosphate oxidases (13, 14). Dual Cancer Research, National Cancer Institute, Frederick, MD 21702 oxidase (Duox)2 is one of the seven members of the Nox Received for publication June 20, 2012. Accepted for publication December 9, 2012. family; although originally described as an H2O2-producing en- This work was supported by the Center for Cancer Research and the Division of Cancer zyme in the thyroid that plays a critical role in thyroid hormone Treatment and Diagnosis, National Cancer Institute, National Institutes of Health; the work was also conducted, in part, under Contract No. HHSN261200800001E. biosynthesis (15), Duox2 also was found in bronchial epithelium The content of this publication does not necessarily reflect the views or policies and throughout the gastrointestinal tract (16, 17). In airway mu- of the Department of Health and Human Services, nor does mention of trade cosal cells, Duox2 plays an important role in the generation of names, commercial products, or organizations imply endorsement by the U.S. H O for host defense against a variety of pathogens (18–20); Government. 2 2 under the stress induced by an infectious agent, Duox2 expression Address correspondence and reprint requests to Dr. James H. Doroshow, Building 31, Room 3A-44, 31 Center Drive, National Cancer Institute, National Institutes of is regulated by several inflammatory stimuli, including IFN-g, Health, Bethesda, MD 20892. E-mail address: [email protected] flagellin, and rhinovirus (16, 20). Duox2-induced ROS also appear The online version of this article contains supplemental material. to play a role in the antibacterial response in the gut (21, 22). Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; ChIP, chromatin However, the expression of Duox2 is significantly increased in immunoprecipitation; CM-H2-DC-FDA, 29,79-dichlorodihydrofluorescein diacetate; human colon biopsies, as well as in isolated intestinal epithelial Duox, dual oxidase; DuoxA2, dual oxidase maturation factor 2; Nox, NADPH oxi- dase; PDTC, pyrrolidinecarbodithioic acid, ammonium salt; ROS, reactive oxygen cells, from patients with inflammatory bowel disease (both species; RT, room temperature; siRNA, small interfering RNA. Crohn’s disease and ulcerative colitis) compared with healthy

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201725 2 INDUCTION OF DUOX2 BY IFN-g AND LPS IN PANCREATIC CANCER

control subjects (21, 23), suggesting that an unchecked ROS re- (CM-H2-DC-FDA) was obtained from Molecular Probes (Carlsbad, CA). sponse to pathogens could contribute to the tissue injury observed 1-Pyrrolidinecarbodithioic acid, ammonium salt (PDTC) (cat. no. 548000) in these chronic inflammatory disorders. and BAY11-70829 (cat. no. 196870) were from EMD Chemicals (Gibbs- town, NJ). MTT (cat. no. M2128) was from Sigma-Aldrich. Our mouse Previous work from our laboratory revealed that the proin- anti-human Duox mAb, S-12, was developed by Creative Biolabs (Port flammatory cytokine IFN-g initiates a Duox2-induced ROS cas- Jefferson Station, NY) and characterized as described previously (24). cade in human pancreatic cancer cells (24). Furthermore, several Although the pancreatic cancer cell lines used for these experiments do not recent studies demonstrated that proinflammatory components of contain measurable Duox1 mRNA, because the Ab cross-reacts with Duox1, we have referred to the protein that it detects as “Duox.” The three the bacterial cell wall, including LPS, mediate Nox-dependent human pancreas tissue microarrays (BIC14011, PA485, PA1001) were ROS generation during the inflammatory response in the airway obtained from US Biomax (Rockville, MD). and gastrointestinal tract, in part due to direct interactions between Cell lines, cell culture, and mice members of the Nox family and TLR4, the critical downstream target that recognizes LPS from Gram-negative bacteria (25, 26). The human pancreatic cancer cell lines BxPC-3 (cat. no. CRL-1687), AsPC- TLR4-related signaling was recently suggested to play a role in 1 (cat. no. CRL-1682), and MIA-PaCa (cat. no. CRL-1420) were obtained the pathogenesis of acute pancreatitis in model systems, as well as from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium (cat. no. SH30255.01; HyClone) with 1% in the clinic (27–29). Because LPS-related TL4 signaling was also pyruvate and 10% FBS. CFPAC-1 pancreatic cancer cells (cat. no. CRL- shown to play a critical role in modulating the invasive potential 1918) were also obtained from the American Type Culture Collection of human pancreatic cancer lines (30), as well as the transition from and were cultured in IMDM with 10% FBS. The identity of each cell line pancreatic inflammation to pancreatic cancer in genetically engi- was confirmed using Identifiler STR genotyping (Applied Biosystems). For starvation conditions, cells were propagated overnight in the same medium neered mouse models (31), we sought to determine whether LPS, without FBS. We used starvation conditions because, although the induction Downloaded from alone or in combination with IFN-g, might regulate Duox2- of Duox2 expression by IFN-g was observed under both serum-containing mediated ROS generation in pancreatic cancer cells. and serum-starved conditions, Duox2 induction was stronger after star- Hence, in this study, we evaluated the effects of IFN-g and LPS vation. Cells were cultured in a humidified incubator at 37˚C in an at- on Duox2 expression and function, as well the mechanism(s) by mosphere of 5% CO2 in air. In control experiments, we found that the basal levels of expression of all of the Nox homologs (Nox1-5, Duox1, Duox2) which these two proinflammatory agents regulate Duox2 levels in in each of the pancreatic cancer cell lines used for these studies were very human pancreatic cancer cell lines. We found that, although both low, often at or near the lower limit of detection of our real-time RT-PCR http://www.jimmunol.org/ agents significantly increase Duox2 expression individually, the assays (data not shown). All mice (female athymic nu/nu NCr) used in the combination synergistically enhances the expression of Duox2 study were obtained from the Animal Production Program (Frederick National Laboratory for Cancer Research,Frederick,MD).National and its associated maturation factor, DuoxA2, leading to a signif- Cancer Institute-Frederick is accredited by American Association for the icant increase in both extracellular and intracellular ROS pro- Accreditation of Laboratory Animal Care International and follows the duction. The dramatic increase in ROS that we observed for the Public Health Service Policy for the Care and Use of Laboratory Animals. combination of LPS and IFN-g depended critically on the up- Animal care was provided in accordance with the procedures outlined in regulation of, and signaling through, the TLR4 pathway, ulti- the Guide for the Care and Use of Laboratory Animals. mately resulting in enhanced binding of NF-kB (p65) to the RNA extraction, cDNA synthesis, and quantitative real-time Duox2 promoter. We also found that ROS production under our RT-PCR assay by guest on September 26, 2021 experimental conditions was sufficient to activate the DNA Total RNA was extracted from IFN-g– or LPS-treated or untreated cells damage-repair pathway while arresting cells in G1, producing a with the RNeasy Mini Kit (cat. no. 74104; QIAGEN), according to the significant increase in apoptosis, and inhibiting cellular prolif- manufacturer’s instructions. Two micrograms of total RNA was used for eration. To determine whether Duox2 might contribute to in- cDNA synthesis, using SuperScript II reverse transcriptase (cat. no. 18080- flammatory states in vivo, we examined its expression both in 044) and random primers (cat. no. 48190-011; Invitrogen) in a 20-ml re- action system, with the following cycles: 25˚C for 5 min, 42˚C for 50 min, tissue specimens from patients with chronic pancreatitis and in and 75˚C for 5 min. After the reaction was complete, the RT-PCR products pancreatic cancer xenografts; in both cases, we found that Duox2 were diluted with diethylpyrocarbonate/H2O to 100 ml for real-time PCR. expression levels were markedly upregulated. These results sug- Real-time RT-PCR was performed on 384-well plates in a 20-ml reaction gest that the pro-oxidant state generated by cytokine-mediated system containing 2 ml diluted cDNA, 1 ml primer mixture, 7 mlH2O, and m 3 Duox2 upregulation could hinder recovery from inflammatory 10 l TaqMan 2 reaction mixture. PCR was carried out under default cycling conditions, and fluorescence was detected with the ABI 7900HT stress-related tissue injury and, thus, may contribute to the de- Sequence Detection System (Applied Biosystems). Triplicate determi- velopment and pathologic behavior of pancreatic cancer cells. nations were performed for each sample that was used for real-time PCR; the mean value was calculated, and the data in the figures represent the results of three independent experiments. Relative was Materials and Methods calculated as the ratio of the target gene to the internal reference gene 6 Materials (b-actin) multiplied by 10 based on Ct values. Recombinant human IFN-g (cat. no. 285-IF) was from R&D Systems. Ab Whole-cell and nuclear extract preparation and Western blot against human b-actin (cat. no. A3853) was acquired from Sigma-Aldrich. analysis Our Stat1 (p84/p91; sc-346) Ab was obtained from Santa Cruz Biotech- nology. Abs against components of the NF-kB complex, including p65 For preparation of whole-cell extracts, cell pellets from BxPC-3, AsPC-1, (cat. no. 6956), IkBa (cat. no. 34812), p-Stat1 (Ser 727) (cat. no. 9177S), and CFPAC-1 cells, treated or not with IFN-g and/or LPS, were lysed with as well as p-Stat1 (Tyr 701) (cat. no. 9167L) and p-Histone gH2AX (Ser 13 RIPA Lysis Buffer (cat. no. 20-188; Millipore, Temecula, CA), with 139) (cat. no. 2577S), were purchased from Cell Signaling Technology. the addition of a phosphatase inhibitor tablet (cat. no. 04-906-837001) and Human TLR4 silencer select small interfering RNA (siRNA; cat. no. a protease inhibitor tablet (cat. no. 11-836-153001; both from Roche). The 4390825), silencer negative control siRNA (cat. no. AM4635), human NE-PER Nuclear and Cytoplasmic Extraction Kit (cat. no. 78833; Thermo Duox2 primer (cat. no. Hs00204187_m1), human DuoxA2 primer (cat. no. Scientific) was used to prepare nuclear extract from cells treated with IFN- Hs01595310_m1), human TLR4 primer (cat. no. Hs00152939_ml), human g and/or LPS. The protein concentrations of both whole-cell and nuclear MyD88 primer (cat. no. Hs00182082_ml), human actin (cat. no. extracts were measured using the BCA Protein Assay Kit (Pierce). Cell Hs99999903_m1), and TaqMan Universal PCR mix (cat. no. 4364340) extracts were mixed with an equal volume of 23 SDS protein gel–loading were from Applied Biosystems (Foster City, CA). The QuikChIP Kit (cat. buffer (cat. no. 351-082-661; Quality Biological), 50 mg whole-cell extract no. 3010K), anti-human TLR4 Ab (cat. no. IMG-6307A), anti-human was loaded onto a 4–20% Tris glycine gel (cat. no. EC6028; Invitrogen), TLR4 FITC Ab, and the Cell Surface TLR Staining Flow Kit (cat. no. and the were separated and electrophoretically transferred to ni- 10099K) were from Imgenex. 29,79-Dichlorodihydrofluorescein diacetate trocellulose membranes using I Blot gel transfer stacks (cat. no. IB 3010- The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. LPS acts synergistically with IFN-g to induce Duox2 and DuoxA2 expression, enhancing both intra- and extracellular production of ROS. (A) Induction of Duox2 expression by exposure to IFN-g (25 ng/ml), LPS (1 mg/ml), or the combination for 24 h; quantitative, real-time RT-PCR analysis of relative Duox2 expression was normalized to b-actin (upper panel). Error bars represent standard deviations; data are from triplicate experiments *p , 0.05, Duox2 expression in IFN-g– or LPS-treated BxPC-3 cells versus solvent-treated controls, ***p , 0.001, LPS and IFN-g pretreatment versus either agent alone. Expression of the Duox protein by Western blot analysis in 50 mg of whole-cell extract from BxPC-3 cells treated with solvent, IFN-g (25 ng/ml), LPS (1 mg/ml), or both IFN-g and LPS for 24 h (lower panel); the Western blot shown is representative of three independent experiments. (B) Expression of both Duox2 and DuoxA2 following treatment with IFN-g (25 ng/ml), LPS (1 mg/ml), or the combination for 24 h was quantitated by real- time RT-PCR normalized to b-actin. Error bars represent standard deviations; data represent the means of three independent experiments. ***p , 0.001, combination of LPS and IFN-g pretreatment versus either agent alone for both Duox2 and DuoxA2. (C) Extracellular H2O2 production was detected using the Amplex Red H2O2/peroxidase assay. BxPC-3 cells were grown in serum-free medium with IFN-g and/or LPS for 24 h. Cells were then collected, and 2 3 104 live BxPC-3 cells were mixed with 100 ml of the Amplex Red reaction mixture containing 50 mM Amplex Red and 0.1 U (Figure legend continues) 4 INDUCTION OF DUOX2 BY IFN-g AND LPS IN PANCREATIC CANCER

01; Invitrogen). The membranes were blocked in 13 TBST buffer with 5% MTT assay for cell proliferation nonfat milk for 1 h at room temperature (RT) and then incubated with BxPC-3 or AsPC-1 cells were seeded in 96-well plates at a density of 1 3 primary Ab overnight in TBST buffer. Membranes were washed three 4 times in 13 TBST buffer and incubated with HRP-conjugated secondary 10 cells/well in 100 ml complete medium overnight. The next day, me- Ab for 1 h at RT with shaking. The Ag–Ab complex was visualized with dium was changed with solvent, and IFN-g (25 ng/ml) or LPS (1 mg/ml) or SuperSignal West Pico Luminol/Enhancer Solution (cat. no. 1856136; both were added. On days 1 and 2 after treatment, cell proliferation was Thermo Scientific). To evaluate Duox2 expression, the whole-cell extract evaluated by the MTT method. In short, the culture medium was removed, was mixed with an equal volume of 23 SDS loading buffer without and 100 ml MTT (0.5 mg/ml) was added to each well. The plates were then boiling; for other proteins, the mixture of cell extract with loading buffer incubated at 37˚C for 45 min. The MTT was removed, and 100 ml MTT was boiled for 5 min. As previously noted (24), Duox2 aggregates when solvent was added to each well. The plates were shaken for 10 min and the whole-cell extract is boiled; the mobility of the Duox2 protein is much read at 570 nm with the plate reader. slower with boiling than without. Cell cycle progression and measurement of apoptosis by Chromatin immunoprecipitation analytical cytometry Chromatin immunoprecipitation (ChIP) assays to detect the binding of the Determination of cell cycle progression was performed using a cell cycle NF-kB p65 subunit to the Duox2 promoter in vivo were carried out with analysis kit (cat. no. L00287; Gen Scrip). BxPC-3 cells grown in complete the QuikChIP Kit (cat. no. 30101K; Imgenex), according to the manu- medium were treated with solvent, 1 mg/ml LPS, 25 ng/ml IFN-g, or both facturer’s protocol. Starved BxPC-3 cells, treated or not with IFN-g and/or LPS and IFN-g for 24 h. Cells were then trypsinized and counted, and 1 3 6 LPS, were cross-linked with 1% formaldehyde, and the cross-linking was 10 cells were washed with 13 PBS and fixed with 70% ethanol at 4˚C for stopped by the addition of glycine (final concentration of 125 mM) to the $2 h or overnight. The fixation solution was removed, and the cells were medium. Cells were then harvested and resuspended in 13 SDS lysis washed once again with 13 PBS. Cells were incubated in 100 ml RNase at buffer. The suspended cells were sonicated to produce 200- to 1000-bp 37˚C for 30 min and then 400 ml propidium iodide was added to each Downloaded from genomic fragments. For immunoprecipitation, NF-kB Ab (cat. no. 6956; sample to stain the cells at 37˚C for an additional 30 min in the dark. Cell Signaling Technology) or normal rabbit IgG Ab (Santa Cruz Bio- Finally, the labeled cells were subjected to analytical cytometry using a BD technology) were used to pull down the cross-linked DNA protein com- FACSCalibur flow cytometer (BD Biosciences). Data from experiments plex. The purified DNA was used as a PCR template to amplify the NF-kB performed in triplicate were analyzed with ModFit v3.0 software. For target sequence in the human Duox2 promoter. The primer sequences were measurement of apoptosis, BxPC-3 cells were propagated in complete as follows: 59-GAGAGGAGCCAGAAGCAGAA-39 and 59-AGACG- medium and then treated with solvent, 1 mg/ml LPS, 25 ng/ml IFN-g,or GAATCCGGAGAGACT-39. The resulting PCR products were 221 bp in both LPS and IFN-g for 72 h. Cells were trypsinized and counted; both length. dead and live cells were collected for our apoptosis assay, which used the http://www.jimmunol.org/ Vybrant Apoptosis Assay Kit #3 (cat. no. V13242; Invitrogen) with a 7- Measurement of intracellular ROS production by flow AAD viability staining solution (cat. no. 420404; BioLegend). Cell la- cytometry beling was carried out according to the manufacturer’s protocol; in brief, 1 3 105 cells were suspended in 100 mlof13 annexin binding buffer to BxPC-3 cells (1 3 106) primed with solvent or with IFN-g and/or LPS which was added 5 ml FITC annexin 5 and 2 ml 7-aminoactinomycin D (7- were suspended in 0.5 ml Krebs buffer containing 7.5 mM of the redox- AAD) viability staining solution. The cells were incubated at RT in the sensitive dye CM-H2-DCF-DA and incubated in the dark for 30 min at dark for 15 min; at the end of that time, 400 ml annexin-binding buffer was 37˚C. After 25 min of incubation, either solvent (DMSO) or 1 mM ionomycin added. The stained cells were analyzed with a BD FACSCalibur flow was added to the cell suspension, and the incubation was continued at 37˚C cytometer as soon as possible thereafter. Data were analyzed with FlowJo for an additional 5 min. The cells were then harvested and resuspended in software. by guest on September 26, 2021 fresh medium without CM-H2-DCF-DA. Fluorescence was recorded on the FL-1 channel of a FACSAria flow cytometer (BD Biosciences, San Jose, Tumor growth, propagation, and sampling CA) and analyzed using FlowJo software (24). Human pancreatic tumor cells for mouse inoculation were used at in vitro passages four to six from cryopreserved cell stocks. Cells (1 3 107 cells/0.1 Extracellular H2O2 measurement using Amplex Red ml/injection) were s.c. inoculated bilaterally into mice (n = 20 mice/tumor The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (cat. no. cell line). This provided a total of 40 potential tumor samples. Two groups A22188; Invitrogen) was used to detect extracellular H2O2 released by IFN- were prepared by randomization on the day of inoculation; 10 mice each g– and/or LPS-activated cells, as described previously (24). BxPC-3 cells were placed into 300- and 500-mg tumor-harvest groups. When tumors were grown in serum-free medium with or without IFN-g and/or LPS for reached the target size, they were resected, cut into quadrants, and flash 24 h; the cells were then washed twice with 13 PBS, trypsinized, and frozen. Flash freezing was accomplished by placing the tumor quadrants dispersed thoroughly. Cells were counted to produce a 20-ml cell sus- into prechilled cryovials, followed by immediate submersion into liquid pension containing 2 3 104 live BxPC-3 cells in 13 Krebs-Ringer phos- nitrogen. All samples were transferred to a 270˚C freezer for holding prior phate glucose buffer. The cells were mixed with 100 ml Amplex Red to processing. For the current experiments, the 300-mg BxPC-3 tumors and reagent containing 50 mM Amplex Red and 0.1 U HRP/ml in Krebs-Ringer the 500-mg AsPC-1 and MIA-PaCa tumors were studied. National Cancer phosphate glucose buffer, with or without 1 mM ionomycin, and incubated Institute-Frederick is accredited by American Association for the Ac- at 37˚C for 60 min. The fluorescence of the oxidized 10-acetyl-3,7-dihy- creditation of Laboratory Animal Care International and follows the U.S. droxyphenoxazine was measured at an excitation wavelength of 530 nm Public Health Service Policy on the Care and Use of Laboratory Animals. and an emission wavelength of 590 nm using a SpectraMax Multiplate All animals used in this research project were cared for and used humanely reader (Molecular Devices, Sunnyvale, CA). H2O2 was quantified with an according to the following policies: The U.S. Public Health Service Policy H2O2 standard curve over a 0- to 2-mM concentration range. Each value in on Humane Care and Use of Animals (1996); the Guide for the Care and the figures represents a mean of quadruplicate samples from 16 readings. Use of Laboratory Animals 8th edition; and the U.S. Government Princi-

of HRP/ml of Krebs-Ringer phosphate buffer with 1 mM ionomycin for 1 h. H2O2 values were calculated by interpolation from a standard curve, using 0–2 mMH2O2. Data are shown as mean 6 SD (n = 16). *p , 0.05, IFN-g or LPS pretreatment versus solvent, ***p , 0.001, combination of IFN-g and LPS versus either agent used independently. (D) Production of intracellular ROS by IFN-g– and/or LPS-treated BxPC-3 pancreatic carcinoma cells as detected by flow cytometry. Starved BxPC-3 cells were treated with solvent, IFN-g (25 ng/ml), LPS (1 mg/ml), or both IFN-g and LPS for 24 h; intracellular ROS production was measured at the end of the incubation by FACS, using the redox-sensitive dye CM-H2-DCF-DA. Ionomycin (1 mM) was added for 5 min following completion of IFN-g and/or LPS treatment, prior to resuspension of the cells in fresh medium and quantitation of fluorescence. The data shown are representative of three independent experiments. (E) Effect of Duox2 siRNA on ionomycin-enhanced extracellular H2O2 production in BxPC-3 cells pretreated with IFN-g or the combination of IFN-g and LPS. The cells were treated with IFN-g or IFN-g and LPS, and H2O2 production was quantitated with ionomycin exposure exactly as described in (C), with the exception that control siRNA or siRNA targeting Duox2 were transfected into BxPC-3 cells 24 h prior to exposure to IFN-g or IFN-g and LPS. Data are mean 6 SD (n = 3). *p , 0.05, IFN-g versus IFN-g and LPS pretreatment following transient transfection with either control or Duox2 siRNA. The Journal of Immunology 5

ples for Utilization and Care of Vertebrate Animals Used in Testing, Re- search, and Training (1985). RNA and protein extraction from tumor xenografts In addition to the methods described above for cell lines, a Polytron System PT 1200E homogenizer equipped with a 5–12-mm-diameter dispersing head (KINEMATICA, Lucerne, Switzerland) was used to homogenize the tumor tissue mixed with a suitable amount of either 13 RIPA buffer for protein extraction or RLT buffer for RNA extraction. A 350-ml volume of tumor homogenate was processed directly for isolation of RNA. For pro- tein lysate preparation, the tumor homogenate in RIPA buffer was centrifuged at 13,000 rpm and 4˚C for 10 min; the supernatant was used for Western blot analysis and to determine protein concentration. Immunohistochemical analysis of Duox expression in pancreatic tissue arrays from patients with chronic pancreatitis Immunohistochemistry using our Duox mAb was performed on three human pancreatic tissue microarrays. Immunohistochemistry staining was performed as follows: slides were deparaffinized with distilled water, and Ag retrieval was performed using DAKO target retrieval solution (cat. no. S1699) for 20 min at 95˚C, followed by 20 min of cooling. Slides were rinsed in distilled water, transferred to TBST buffer (cat. no. S3006; Downloaded from DAKO) for 5 min, and incubated in peroxidase blocking solution (3% H2O2) for 10 min at RT. Duox Ab, at a dilution of 1:250 (diluted in DAKO diluent; cat. no. S0809), was used for 60 min at RT; slides were then rinsed in TBST, and secondary Ab was applied (DAKO EnVision+ Dual Link System-HRP [cat. no. K4063], Ready-To-Use) for 30 min at RT. The slides were rinsed in TBST (three times, 2 min each); 3,39-diaminobenzidine was used for 10 min for visualization, and the slides were rinsed in TBST. http://www.jimmunol.org/ Hematoxylin was used as a counterstain. Positive control tissues (normal human colon and cell pellets) and a negative control reagent (normal mouse IgG1 isotype control, 10 mg/ml for 60 min [cat. no. 550878; BD Bio- sciences], diluted in DAKO diluent [cat. no. S0809]) were also examined. Statistical analysis Results are expressed as the mean 6 SD from at least triplicate experi- ments. Statistical differences between mean values of control and treated samples were assessed using the Student t test; p , 0.05 was considered statistically significant. by guest on September 26, 2021 Results Combination of LPS and IFN-g synergistically induces Duox2 expression and reactive oxygen production BxPC-3 cells were treated with IFN-g and/or LPS to determine the potential of each, as well as the combination, to induce Duox2 expression. LPS alone significantly increased Duox2 mRNA lev- els in BxPC-3 cells, albeit to a lesser degree than IFN-g; however, the combination of LPS and IFN-g increased Duox2 expression ∼6-fold compared with IFN-g alone (p , 0.001; Fig. 1A, upper panel). In control experiments, we found that decreasing the LPS concentration to 100 ng/ml did not alter the synergistic induction of Duox2 expression with IFN-g that we observed; Duox2, rather than Duox1, was the predominant oxidase affected by the com- bination of IFN-g and LPS (Supplemental Fig. 3). Western blot analysis confirmed that LPS- and IFN-g–induced Duox2 mRNA expression correlated well with Duox protein levels (Fig. 1A, lower panel). The simultaneous exposure of cells to both LPS and IFN-g induced a substantially higher level of Duox protein ex- pression than did either LPS or IFN-g (Fig. 1A). In like manner

FIGURE 2. Enhanced TLR4 expression is associated with LPS- and IFN-g–mediated increases in Duox2/DuoxA2 levels in BxPC-3 cells. (A) blot analysis with TLR4-, MyD88-, p-Stat1–, and Stat1-specific Abs. Data BxPC-3 cells were grown in serum-free medium and treated with IFN-g shown are representative of three independent experiments. (C) IFN-g– (25 ng/ml) or LPS (1 mg/ml) or both for 24 h. RNA was then extracted and induced cell surface expression of TLR4 was detected by analytical subjected to real-time RT-PCR to evaluate TLR4/MyD88 expression nor- cytometry. Starved BxPC-3 cells were treated with solvent or IFN-g (25 malized to b-actin. Error bars represent standard deviations; data are from ng/ml) for 24 h and then stained with either FITC-conjugated isotype triplicate experiments (nine readings). (B) Fifty micrograms of whole-cell control or human TLR4-specific Ab; labeled cells were then analyzed by extract from BxPC-3 cells were treated with solvent or exposed to IFN-g flow cytometry. The graph shown is an example taken from three separate (25 ng/ml), LPS (1 mg/ml), or both for 24 h and then subjected to Western experiments. ***p , 0.001, versus solvent alone. 6 INDUCTION OF DUOX2 BY IFN-g AND LPS IN PANCREATIC CANCER

(Fig. 1B), we found that the addition of LPS to IFN-g significantly strated that intracellular ROS levels were increased following increased the expression of the Duox2 maturation factor, DuoxA2, exposure of BxPC-3 cells to LPS and/or IFN-g and that the ROS which plays a critical role in the formation of the active Duox2 levels observed were consistent with the expression levels of membrane oxidase (32). To determine whether these levels of Duox2 and DuoxA2 following exposure to LPS or IFN-g, alone Duox2 and DuoxA2 correlated with functional oxidase activity, or in combination (Fig. 1D). Although intracellular ROS produc- we examined the production of ROS using two techniques. In the tion in BxPC-3 cells following ionomycin treatment was modestly presence of 1 mM ionomycin for 1 h, which is known to activate increased by LPS alone and was increased to a greater degree by Duox 2 (13), BxPC-3 cells previously exposed for 24 h to IFN-g IFN-g, exposure to the combination of LPS and IFN-g resulted in and/or LPS produced significant amounts of extracellular H2O2 substantially greater ROS production than did exposure to either (Fig. 1C), as detected with Amplex Red reagent; cotreatment with agent alone (Fig. 1D). Finally, we found that ionomycin-enhanced LPS and IFN-g resulted in significantly more extracellular H2O2 H2O2 production by BxPC-3 cells exposed to LPS and IFN-g production than did exposure to either agent alone. To confirm our could be decreased significantly following pretreatment with results obtained with Amplex Red, we performed flow cytometric Duox2-specific siRNA (Fig. 1E). Taken together, these data are quantitation of intracellular ROS production using the redox- consistent with the synergistic induction of functional Duox2 and sensitive dye CM-H2-DCF-DA. These studies clearly demon- DuoxA2 by the combination of LPS and IFN-g. Downloaded from

FIGURE 3. Silencing TLR4 attenuates IFN-g– and http://www.jimmunol.org/ LPS-mediated enhancement of TLR4 and Duox2/ DuoxA2 expression in BxPC-3 cells. (A) Inhibition of TLR4 expression by TLR4 siRNAs. Control siRNA or two human TLR4-specific siRNAs targeting dif- ferent domains of TLR4 were transiently transfected into BxPC-3 cells; 24 h following transfection, cells were incubated in serum-free medium with or with- out IFN-g (25 ng/ml) or LPS (1 mg/ml) for another 24 h. RNA was then extracted and subjected to real- time RT-PCR, and relative TLR4 levels were nor- by guest on September 26, 2021 malized to b-actin. Error bars represent SD; data are from triplicate samples. (B) Silencing TLR4 produced no effect on IFN-g–induced Stat1 expression. The identical RNA samples used in (A) were subjected to real-time RT-PCR assay with human Stat1-specific primers, and Stat1 expression relative to b-actin was calculated. (C) Silencing of TLR4 attenuated IFN-g– and LPS-induced Duox2 expression. The same RNA samples used for the experiments shown in (A) were subjected to real-time RT-PCR using human Duox2- specific primers, and Duox2 expression relative to b-actin was calculated. (D) Silencing TLR4 attenu- ated IFN-g– and LPS-induced DuoxA2 expression. The RNA samples used for the experiments in (A) were subjected to real-time RT-PCR assay, using human DuoxA2 primers; DuoxA2 expression relative to b-actin was calculated. (E) Control or TLR4-spe- cific siRNAs were transiently transfected into BxPC- 3 cells. At 24 h after transfection, cells were incu- bated in serum-free medium with or without IFN-g (25 ng/ml) or LPS (1 mg/ml) for a subsequent 24 h, and 50 mg of whole-cell extract was subjected to Western blot analysis, using specific Abs as indicated. These results are representative of three separate experiments. ***p , 0.001, versus BxPC-3 cells treated with a control siRNA. The Journal of Immunology 7

IFN-g and LPS upregulate TLR4 and MyD88 in association TLR4, but not MyD88, levels further (Fig. 2A). Western blot with enhanced Duox2 expression levels analysis confirmed that the increase in IFN-g–induced TLR4 and It was reported that exposure to IFN-g augments the expression of MyD88 mRNA correlated with an increase in protein expression; TLR4 and its accessory components, MD-2 and MyD88, in human we also found, as previously described (24), that treatment of monocytes and macrophages (33). In those experiments, exposure BxPC-3 cells with IFN-g enhanced the phosphorylation of Stat1 of IFN-g–primed monocytes to LPS enhanced the phosphoryla- at tyrosine 701 (Fig. 2B). Finally, we evaluated the surface expres- tion of IRAK, NF-kB DNA-binding activity, and TNF-a and IL- sion of TLR4 after IFN-g exposure by flow cytometry using an 12 production. Furthermore, Abreu et al. (34) reported that in- FITC-labeled human TLR4 Ab. BxPC-3 cells demonstrated ex- testinal epithelial cells expressed low levels of TLR4 and MD-2 pression of TLR4 on the cell surface that was enhanced by IFN-g, and were hyporesponsive to LPS; however, in the same cells, the resulting in a substantial right shift of the fluorescent signal (Fig. 2C). proinflammatory cytokines IFN-g and TNF-a increased TLR4 and MD-2 expression and sensitized epithelial cells to LPS-dependent Silencing of TLR4 attenuates IFN-g– and LPS-mediated IL-8 secretion. We sought to determine whether our observed induction of TLR4 and Duox2/DuoxA2 expression synergistic induction of Duox2 by LPS and IFN-g occurred by the We used RNA interference to further define the role of TLR4 in same mechanism. We used real-time RT-PCR to evaluate the ex- the synergistic induction of Duox2 and DuoxA2 by LPS and IFN-g pression of TLR4 and its adaptor protein MyD88 after exposure in BxPC-3 cells. Control siRNA or two distinct human TLR4- of BxPC-3 cells to solvent, LPS, and/or IFN-g. IFN-g induced specific siRNAs targeting different domains of TLR4 were tran- a significant, ∼3–4-fold increase in TLR4 and MyD88 expression siently transfected into BxPC-3 cells. At 24 h following trans- in BxPC-3 cells, and the combination of LPS and IFN-g increased fection, cells were cultured in serum-free medium with LPS and/ Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. LPS does not enhance IFN-g–induced Duox2 expression in AsPC-1 human pancreatic cancer cells. (A) Starved AsPC-1 cells were treated with IFN-g (25 ng/ml) or LPS (1 mg/ml) for 24 h; 2 mg of total RNA was subjected to real time RT-PCR, and Duox2 expression relative to b-actin was determined. Error bars represent SD; data are from triplicate samples. (B) TLR4 expression was determined using real-time RT-PCR with the same RNA as shown in (A). Human TLR4-specific primers were used for PCR. (C) LPS activated the NF-kB–signaling pathway in BxPC-3 cells. BxPC-3 cells grown in serum-free medium were treated with solvent or IFN-g (25 ng/ml) for 24 h and then incubated with or without LPS (1 mg/ml) for 1 h; 50 mg of whole-cell extract was subjected to Western blot analysis. These experiments were performed in triplicate. (D) LPS did not enhance IFN-g–mediated Duox2 ex- pression in AsPC-1 cells. Starved AsPC-1 cells were treated with IFN-g (25 ng/ml) or LPS (1 mg/ml) for 24 h, and 50 mg of whole-cell extract was subjected to Western blot analysis, using specific Abs as indicated. The results are representative of triplicate experiments. (E) LPS did not activate the NF-kB–signaling pathway in AsPC-1 cells. AsPC-1 cells grown in serum-free medium were treated with solvent or IFN-g (25 ng/ml) for 24 h and then incubated with or without LPS (1 mg/ml) for 1 h; 50 mg of whole-cell extract was then subjected to Western blot analysis. The data shown are representative of three identical experiments. (F) Effect of LPS on p65 nuclear accumulation in BxPC-3 and AsPC-1 cells exposed to IFN-g. BxPC-3 and AsPC-1 cells were treated as described in (A), and 20 mg of nuclear extract was subjected to Western blot analysis to determine Stat1, IRF-1, and p65 distribution in the nucleus. Lamin A/C was used as a loading control for nuclear protein. The data are representative of experiments performed in triplicate. ***p , 0.001, versus AsPC-1 cells treated with solvent. 8 INDUCTION OF DUOX2 BY IFN-g AND LPS IN PANCREATIC CANCER or IFN-g for another 24 h. Cells were then collected and subjected extracts from AsPC-1 cells that had been treated for 24 h with LPS to RNA extraction and real-time RT-PCR. Human TLR4 siRNAs and/or IFN-g were subjected to Western blot analysis. As shown were found to specifically suppress IFN-g– and LPS-induced in Fig. 4D, IFN-g activated Stat1 signaling in this cell line and TLR4 expression (Fig. 3A) but had no inhibitory effect on IFN- augmented Duox expression. IFN-g treatment also induced TLR4 g–induced Stat1 expression (Fig. 3B). Human TLR4 siRNAs also expression; however, unlike BxPC-3 cells, cotreatment with LPS significantly attenuated the expression of IFN-g– and LPS- did not result in a further increase in Duox protein levels. We next induced Duox2 (Fig. 3C) and DuoxA2 (Fig. 3D), whereas con- compared NF-kB signaling in the two cell lines after priming the trol siRNAs had no inhibitory effect on the expression of IFN- cells with either solvent or IFN-g for 24 h and then exposing the g– and LPS-induced TLR4 or Duox2/DuoxA2. Western blot cells to LPS for 1 h. In BxPC-3 cells, priming the cells with IFN-g analysis confirmed that TLR4 siRNA had an inhibitory effect on and treating for 1 h with LPS resulted in IkBa degradation (Fig. LPS- and IFN-g–induced TLR4 protein expression (Fig. 3E). Si- 4C, compare lanes 1 and 4). In contrast, for AsPC-1 cells, expo- lencing of TLR4 also decreased LPS- and IFN-g–induced Duox sure to LPS plus IFN-g did not result in IkBa degradation (Fig. protein expression (Fig. 3E, lanes 4 and 8). 4E). Finally, we examined nuclear extracts prepared from BxPC-3 and AsPC-1 cells that had been primed with either solvent or IFN- Intact NF-kB signaling is required for LPS- and g for 24 h and then treated with LPS for 1 h. In both cell lines, IFN-g–induced Duox2 expression IFN-g priming resulted in a similar level of nuclear accumulation To expand our results with BxPC-3 cells, the AsPC-1 human of Stat1 and IRF-1 (Fig. 4F), which was not dependent on LPS pancreatic cancer cell line was also exposed to IFN-g; as dem- treatment. However, results for the transcription factor NF-kB onstrated in Fig. 4A, these cells also respond to IFN-g by in- differed in the two cell lines. In BxPC-3 cells, LPS exposure alone Downloaded from creasing the expression of Duox2. We also sought to determine resulted in a clear nuclear translocation of NF-kB; priming with whether LPS and IFN-g act synergistically to induce Duox2 ex- IFN-g prior to treatment with LPS resulted in further enhancement pression in AsPC-1 cells as they do in the BxPC-3 line. Starved of the nuclear accumulation of NF-kB. However, in AsPC-1 cells, AsPC-1 cells were treated with solvent or with LPS (1 mg/ml) and/ the nuclear distribution of NF-kB was not influenced by either or IFN-g (25 ng/ml), and real-time RT-PCR was used to evaluate LPS or IFN-g treatment (Fig. 4F). To evaluate the generality of

TLR4 and Duox2 expression. Although treatment of AsPC-1 cells our finding that NF-kB signaling could play an important role in http://www.jimmunol.org/ with IFN-g alone induced a significant level of Duox2 expres- regulating the expression of Duox2 in human pancreatic cancer sion, in marked contrast to our results in BxPC-3 cells, exposure cells, we also evaluated the effects of IFN-g and LPS on CFPAC-1 to LPS plus IFN-g did not result in a further significant increase in human pancreatic cancer cells (Supplemental Fig. 1). In this line, Duox2 expression (Fig. 4A). Furthermore, IFN-g, either alone or similar to our observations with BxPC-3 cells, we found that ex- in combination with LPS, increased TLR4 expression by ∼4-fold posure to IFN-g and LPS produced a more-than-additive effect on over the levels observed with solvent or LPS (Fig. 4B). Whole-cell Duox2 expression at both the RNA and protein levels that was by guest on September 26, 2021

FIGURE 5. Two independent NF-kB inhibitors attenuate LPS-mediated enhancement of Duox2 expression in IFN-g–primed BxPC-3 cells, and ChIP detects binding of NF-kB p65 to the Duox2 promoter in BxPC-3 cells. (A) BxPC-3 cells were grown in serum-free medium and pretreated with DMSO as the vehicle or with the NF-kB inhibitors PDTC (100 mM) or BAY 11-7082 (10 mM) for 30 min. Cells were then stimulated with IFN-g (25 ng/ml) or LPS (1 mg/ml) for 24 h, and RNA was extracted and subjected to real-time RT-PCR to determine Duox2 expression relative to b-actin. Error bars represent SD; data are from triplicate experiments (nine readings for each experimental condition). (B) ChIP assay detects NF-kB p65 binding to the Duox2 promoter. Starved BxPC-3 cells were treated with solvent or IFN-g (25 ng/ml) for 24 h and then exposed to LPS (1 mg/ml) for 1 h. Cells were then harvested and used for ChIP. Input lanes verify that equal amounts of DNA were used for the initial immunoprecipitation. Control IgG- and p65-specific Abs were used to pull down DNA, which was then extracted and used for PCR with primers spanning the potential NF-kB binding site in the Duox2 promoter. The resulting PCR products were separated on a 2% agarose gel and visualized with ethidium bromide staining. The experiment was performed in triplicate. ***p , 0.001, versus IFN-g and LPS exposure without inhibitor pretreatment. The Journal of Immunology 9 accompanied by activation of TLR4 and was associated with degradation of IkBa (Supplemental Fig. 1). Furthermore, nuclear accumulation of NF-kB following LPS and IFN-g exposure re- sembled our findings in BxPC-3, and not in AsPC-1, cells (Sup- plemental Fig. 2). These results suggest that an intact NF-kB– signaling pathway is necessary for the synergistic induction of Duox2 by LPS and IFN-g in human pancreatic cancer cells. Inhibition of NF-kB diminishes LPS- and IFN-g–induced Duox2 expression in BxPC-3 cells To investigate further whether LPS- and IFN-g–mediated induction of Duox2 depended on NF-kB activation, we exposed BxPC-3 cells to two NF-kB inhibitors, PDTC (100 mM) and BAY 11-7082 (10 mM), for 30 min prior to LPS and/or IFN-g. Both inhibitors significantly decreased LPS- and IFN-g–mediated Duox2 induction (Fig. 5A), indicating that activation of NF-kB is required for Duox2 induction by LPS and IFN-g. Neither NF-kB inhibitor, over the time course and concentrations tested, was cytotoxic, as measured by exclusion of trypan blue dye (data not shown). Downloaded from NF-kB p65 binds to the human Duox2 promoter Having confirmed that IFN-g exposure can upregulate TLR4 and MyD88 expression in BxPC-3 cells and is associated with a more- than-additive effect on Duox2 expression when combined with

LPS, as well as accumulation of NF-kB p65 in the nucleus, we http://www.jimmunol.org/ sought to determine whether a component of the NF-kB complex could bind to the human Duox2 promoter. We also wished to examine whether such binding would be enhanced by cotreatment with LPS and IFN-g over that seen with either LPS or IFN-g treatment alone. ChIP was used to investigate binding of the NF- kB p65 subunit in vivo under various experimental conditions. Using the TRANSFAC search program (35), we identified a ca- nonical NF-kB binding site within 3 kb of the human Duox2 promoter between 22771 and 22763 bp from the transcription by guest on September 26, 2021 start site. The sequence was 59-GGGAATTCCC-39, which exactly matched the known NF-kB consensus motif (59-GGGRNNTYCC-39) provided by TRANSFAC. Therefore, PCR primers were designed on the basis of the DNA sequence surrounding the potential NF- kB binding site. Equal amounts of DNA were used for the initial immunoprecipitation (Fig. 5B, top panel). Control IgG Ab pro- FIGURE 6. Comparison of the effects of IFN-g and LPS on extracel- duced a very weak background PCR signal, indicating immu- lular H2O2 production and cell growth inhibition in BxPC-3 and AsPC-1 noprecipitation, as well as that PCR reactions were specific (Fig. pancreatic cancer cells. Extracellular H2O2 was detected using the Amplex 5B, middle panel). With a p65-specific Ab (Fig. 5B, bottom panel), Red assay. BxPC-3 or AsPC-1 cells were grown in serum-free medium LPS and IFN-g treatment produced a clear PCR product; further- treated with solvent, LPS (1 mg/ml), IFN-g (25 ng/ml), or both LPS and more, cotreatment with LPS and IFN-g produced a much stronger IFN-g for 24 h. The Amplex Red assay was then conducted as described in PCR signal that was indicative of greater p65 binding to the en- Fig. 1C for both BxPC-3 and AsPC-1 cells to detect extracellular H2O2 dogenous human Duox2 promoter. production at different times. (A) Reaction buffer contained DMSO as the vehicle. H2O2 was measured for 4 h in the four experimental groups. The 6 LPS and IFN-g treatment leads to extracellular H2O2 data are expressed as the mean SD for multiple independent experiments accumulation, inhibition of growth and cell cycle progression, (n = 16 for each time point evaluated). (B) Inclusion of 1 mM ionomycin in and induction of apoptosis and DNA damage in human the reaction buffer resulted in enhanced H2O2 generation. Data are from 6 C pancreatic cancer cells multiple experiments and are expressed as means SD (n = 16). ( )H2O2 production by AsPC-1 cells was determined exactly as described for the To examine potential consequences of LPS- and IFN-g–mediated BxPC-3 line in the absence of added ionomycin. The data shown are mean upregulation of Duox2 and DuoxA2, we examined the time course values from multiple determinations (n = 16). (D)H2O2 production by of extracellular H2O2 accumulation following a variety of stimu- AsPC-1 cells was examined as described in Fig. 1C in the presence of lation conditions. Surprisingly, in the absence of ionomycin stim- 1 mM ionomycin. The data represent multiple experiments with n = 16. (E) Cell growth was inhibited by exposure to IFN-g and the combination of ulation, extracellular H2O2 production by BxPC-3 cells steadily increased over time; after 4 h of incubation, extracellular H O IFN-g and LPS in BxPC-3 cells. BxPC-3 cells grown in serum-free me- 2 2 dium were treated with solvent, LPS (1 mg/ml), IFN-g (25 ng/ml), or both production for cells that had been exposed for 24 h previously to LPS and IFN-g for 1 or 2 d. MTT assays were then used to evaluate cell IFN-g or the combination of LPS plus IFN-g continued to gen- proliferation. Data are expressed as mean 6 SD for multiple experiments erate H2O2 at substantial levels (Fig. 6A). When ionomycin was (n = 16). (F) Neither IFN-g alone nor the combination of IFN-g and LPS added to the reaction system, the differences among the treatment significantly inhibited cell growth in the AsPC-1 cell line under experi- conditions became more pronounced (Fig. 6B); cells primed with mental conditions identical to those used for BxPC-3 cells. ***p , 0.001, LPS plus IFN-g produced the highest amounts of extracellular versus cells exposed to vehicle alone. 10 INDUCTION OF DUOX2 BY IFN-g AND LPS IN PANCREATIC CANCER

H2O2, consistent with their expression levels of Duox2 and sustained intra- and extracellular ROS production mediated by DuoxA2. In contrast, consistent with the lower levels of Duox2 LPS- and IFN-g–induced Duox2 expression in BxPC-3 cells may expression measured in AsPC-1 cells following treatment with be sufficient (at least compared with the AsPC-1 line) to affect IFN-g and/or LPS, the production of H2O2 was lower in these tumor cell proliferation. To evaluate potential mechanisms of cell cells (Fig. 6C) and was enhanced to a much lesser degree by the death, we examined the effects of IFN-g and LPS on cell cycle presence of the calcium ionophore (Fig. 6D). We next measured progression and apoptosis in BxPC-3 cells. As shown in Fig. 7A, the effects of LPS and IFN-g on cell growth in BxPC-3 and AsPC- exposure of BxPC-3 cells to IFN-g and LPS produced a profound 1 cells using the MTT assay (Fig. 6E, 6F). On day 1, there was no inhibition of cell cycle progression at G1 that exceeded that of significant difference among the various treatment groups. On day IFN-g alone and which is consistent with an effect of H2O2 (36); 2, LPS alone still demonstrated no inhibitory effect on cell pro- the G1 arrest was associated with a marked degree of both apo- liferation; in contrast, IFN-g alone significantly inhibited the ptotic cell death and loss of cell membrane permeability (Fig. 7B). growth of BxPC-3 cells. The combination of LPS and IFN-g Finally, to examine the possibility that Duox2-related reactive resulted in .50% retardation of cell growth by day 2 of exposure. oxygen metabolism might contribute to enhanced genetic insta- In comparison, IFN-g induced both Duox2 and DuoxA2 expres- bility of our pancreatic cancer cells, we evaluated the effect of sion in AsPC-1 cells to a lesser degree than for the BxPC-3 line; IFN-g and LPS on the activation of phosphorylated gH2AX, IFN-g alone or combined with LPS did not have an inhibitory a biomarker for DNA damage and repair (37). As shown in Fig. effect on AsPC-1 cell growth (Fig. 6F). These data suggest that the 7C, we found that both IFN-g alone or the IFN-g and LPS com- Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. Effects of IFN-g, LPS, or the combination of IFN-g and LPS on cell cycle progression, apoptosis, and DNA damage in BxPC-3 cells. (A)BxPC- 3 cells that had been grown in complete media were treated with solvent, LPS (1 mg/ml), IFN-g (25 ng/ml), or both LPS and IFN-g for 24 h. Cells were collected, fixed with 70% ethanol, stained with propidium iodide, and subjected to analytical cytometry. The data were analyzed with ModFit LT software. (B) To examine the effects of LPS and/or IFN-g on cell death in the BxPC-3 line, cells were exposed to each agent individually and in combination for 72 h; live and dead (floating) cells were collected for estimation of the degree of apoptosis using the Vybrant Apoptosis Kit #3 with 7-AAD viability staining solution. Labeled cells were analyzed by flow cytometry. Dead cells that cannot exclude 7-AAD were detected in the far red spectral range, depicted on the y-axis; apoptotic cells labeled with FITC conjugated to annexin V are shown with green fluorescence on the x-axis. (C)IFN-g and LPS produce DNA double-strand breaks in BxPC-3 cells detected by an increase in phosphorylated gH2AX protein. Cells were treated with IFN-g or the combination of IFN-g and LPS for

72 h; the thiol-containing ROS scavenger N-acetyl-L-cysteine (NAC) or the cell-permeable H2O2-detoxifying enzyme PEG-catalase (PEG-CAT) was ad- ministered 30 min prior to the initiation of IFN-g or IFN-g plus LPS exposure to evaluate the effect of scavenging ROS on the DNA damage response. Western blot analysis was performed with 50 mg of cell lysate using the specific Abs, as indicated. The results shown are typical of three identical experiments. The Journal of Immunology 11 bination induced a marked DNA damage response in BxPC-3 tumor in vivo as a xenograft (Fig. 8C, upper panel). Tumors of cells. Furthermore, the gH2AX signal was decreased by pre- 500 mg, rather than 300 mg, were used to study the AsPC-1 and treatment of the BxPC-3 cells with either the ROS scavenger MIA-PaCa xenografts to optimize the chance for upregulation of N-acetyl-L-cysteine or by a formulation of the H2O2 detoxifying Duox2. Western blot analysis from lysates prepared from the same enzyme catalase (PEG-catalase) that crosses the tumor cell outer tumors (that had been split into quarters) used for RT-PCR con- membrane. These studies indicate that ROS produced by IFN-g in firmed the changes in expression that we found at the mRNA level combination with LPS may produce DNA damage that activates (Fig. 8A–C, lower panels). a DNA repair response measurable by the expression of gH2AX. Duox expression is increased in patients with chronic Growth of human pancreatic cancer cells as xenografts is pancreatitis associated with increased Duox2 expression To evaluate the expression of Duox earlier in the process of In our current experiments, as well as our previous study (24), we pancreatic carcinogenesis, we performed immunohistochemical found that Duox2 expression in human pancreatic cancer cells analyses using three human pancreatic tissue arrays that included in culture is very low in the absence of cytokine exposure, and normal pancreatic tissue from 10 patients (18 spots) and chronic certain pancreatic lines (e.g., MIA-PaCa cells) are unresponsive to pancreatitis specimens from 48 patients (49 spots). The specimens IFN-g. We hypothesized that it might be possible to evaluate the of normal pancreas had a low-level, diffuse, cytoplasmic signal that relevance of our current investigations to the setting of tumor cell was most noticeable within pancreatic islets (Fig. 9A). None of the growth in vivo by examining whether the extracellular milieu of normal pancreas specimens had evidence of membrane staining. pancreatic tumor cells growing as xenografts could provide an The majority of the pancreatitis specimens (34/48) had multifocal, Downloaded from environment that was sufficiently proinflammatory to alter Duox2 markedly increased Duox staining in both cytoplasm and mem- expression in the absence of treatment with an exogenous cyto- brane that was most noticeable in duct(ule)s (Fig. 9B). Minimal kine. As shown in Fig. 8, we found that during the first passage of inflammation and low numbers of ducts, as well as sampling vari- BxPC-3 or AsPC-1 cells in athymic mice, when tumors reached ability, were considered the probable reasons for negative results in 300 or 500 mg in size, respectively, Duox2 expression increased some of the pancreatitis cases (data not shown). Increased staining

dramatically compared with the expression levels in the cells in patients with pancreatitis was often closely associated with areas http://www.jimmunol.org/ immediately prior to xenografting. Furthermore, for both the of inflammatory cell infiltrates (Fig. 10). BxPC-3 and AsPC-1 lines, Duox2 expression in vivo approxi- mated that following exposure to IFN-g for 24 h in vitro (Fig. 8A, Discussion 8B, upper panels). In contrast, the level of Duox2 in MIA-PaCa Oxidative stress plays a critical role in modulating the innate cells, which is at the lower limit of detection by RT-PCR and does immune response to inflammatory stimuli (38, 39). Further, there is not respond to IFN-g, did not change following growth of the increasing evidence that the source of at least some of the ROS by guest on September 26, 2021

FIGURE 8. A single in vivo passage as a tumor xenograft significantly increases the expression of Duox2 in BxPC-3 and AsPC-1, but not MIA-PaCa, pancreatic cancer cells. BxPC-3, AsPC-1, and MIA-PaCa cells were grown as human tumor xenografts directly from cultured cell lines, as described in Materials and Methods.(A and B) For BxPC-3 and AsPC-1 cells, Duox2 expression was significantly higher in tumor xenografts than in either cell line examined by real time RT-PCR immediately prior to implantation (upper panels)(n = 6 or 7 individual xenografts for AsPC-1 or BxPC-3 cells, re- spectively). Western blot analysis confirmed the increase in Duox expression for the individual tumors (lower panels). (C) Duox2 expression examined by RT-PCR in MIA-PaCa cells is at the lower limit of detection of our assay; therefore, 24-h exposure to IFN-g (25 ng/ml) in culture cannot be said to change

Duox2 levels. Furthermore, based on Ct values, Duox2 levels in MIA-PaCa cell xenografts are also at the lower limit of detection of our assay. Duox expression at the protein level could not be demonstrated for MIA-PaCa cells grown as xenografts. Lysates from BxPC-3 cells were used as simultaneously evaluated positive controls. ***p , 0.001, pretreatment Duox2 levels in BxPC-3 or AsPC-1 cells in tissue culture versus xenografts or cells exposed to 25 ng/ml IFN-g in culture for 24 h. 12 INDUCTION OF DUOX2 BY IFN-g AND LPS IN PANCREATIC CANCER Downloaded from http://www.jimmunol.org/ FIGURE 9. Immunohistochemical analysis demonstrates increased ex- pression of Duox in patients with chronic pancreatitis. (A) Normal human FIGURE 10. Increased expression of Duox in human cases of pancre- pancreas. Low-level, diffuse, cytoplasmic expression of Duox (light brown atitis is associated with areas of inflammation. (A) Normal human pan- stippling) was most noticeable in islets. This diffuse background staining creas. Minimal staining is evident by immunohistochemistry. (B–D) Three was routinely observed in samples evaluated from 10 patients. (B) Human cases of pancreatitis. Increased staining intensity is multifocal, with pro- pancreas with pancreatitis. Duox expression was increased, with notable pensity toward areas of inflammatory cell infiltration. Variation in size of membrane and cytoplasmic staining, particularly in ductal structures. spots is due to different-sized microarrays (48 versus 100 spots). Original Similar, increased intensity of cytoplasmic and membrane staining for magnification 35. IN, Inflammation; L, islet. Duox was present in 34 of 48 cases of pancreatitis on the tissue micro- arrays evaluated. D, Ductule; IN, inflammation; L, islet. by guest on September 26, 2021 We found that the combination of IFN-g and LPS produced a substantially greater-than-additive effect on Duox2 expression that accompany acute and chronic inflammation in many organs is in BxPC-3 (Fig. 1) and CFPAC-1 (Supplemental Fig. 1), but not one or more epithelial members of the Nox family of oxidases (in AsPC-1 (Fig. 4), pancreatic cancer cells. The major increase in addition to the Nox2 found in granulocytes and macrophages) (3, expression of Duox2 and DuoxA2 that we observed was accom- 4, 40). Thus, in the experiments reported in this article, we sought panied by a significant enhancement of functional oxidase activity to evaluate in pancreatic cancer cells the mechanism(s) by which measured by extracellular H2O2 formation and intracellular ROS proinflammatory stimuli regulate the expression of Duox2, the production (Fig. 1). Although IFN-g increased the expression of Nox family member that supplies a substantive amount of the activated Stat1 to a similar degree in all three cell lines, the addition H2O2 used for host defense by airway mucosal cells, as well as of LPS did not lead to any further change in Duox2 levels in AsPC- organs of the gastrointestinal tract (17, 41). 1 cells compared with the BxPC-3 and CFPAC-1 lines (Fig. 4). In a previous study, we demonstrated that IFN-g upregulated The divergent responses of these cell lines to cotreatment with both Duox2 and its critical maturation factor DuoxA2 in a Stat1- LPS and IFN-g prompted us to examine the underlying mecha- dependent fashion that involved both the Jak and p38-MAPK nisms that might explain these differences. Under baseline con- pathways (24). Specific binding of Stat1 to the Duox2 promoter ditions, NF-kB is sequestered in the cytoplasm by the inhibitory plays an essential role in the upregulation of Duox2 in BxPC-3 activity of IkBa. The engagement of LPS to the TLR4 receptor human pancreatic cancer cells, as well as the subsequent Duox2- results in the activation of IkB kinases. Activated IkBkinases related production of a substantive flux of H2O2. It was observed phosphorylate IkBa, leading to its ubiquitination and subsequent that pancreatic cancer lines, like certain other epithelial tumors, degradation. Once IkBa is degraded, NF-kBisreleasedand express TLRs (30) and are capable of signal transduction through translocates to the nucleus where it can bind to DNA sequences TLR-relatedpathwayspreviouslythoughttobeanexclusive known as kB sites, resulting in the transcriptional activation of characteristic of immune cells (42). Furthermore, Ochi et al. (31) many inflammation-related (43). A critical role for the NF- recently reported that LPS accelerated tumorigenesis in the pan- kB pathway in the explanation of our findings is supported by the creas and that inhibition of TLR4 signaling significantly decreased observation that pretreatment with inhibitors of NF-kB signaling, the development of inflammation-related pancreatic tumors PDTC and BAY117082, significantly decreased Duox2 expression in vivo. These studies support our evaluation of the effect of LPS following exposure to IFN-g and LPS (Fig. 5A). Furthermore, as alone and in combination with IFN-g on the formation of ROS by shown in Fig. 4 and Supplemental Fig. 2, following IFN-g priming, cancer cells of pancreatic origin, as well as our examination of exposure to LPS in BxPC-3 and CFPAC-1 cells leads to degra- Duox2 expression during the growth of pancreatic tumor models dation of IkBa and substantial translocation of NF-kB (p65) to in vivo and in the process of pancreatic inflammation in man. the nucleus, whereas translocation of NF-kB (p65) to the nucleus The Journal of Immunology 13 is minimal in AsPC-1 cells under the same experimental conditions. patients with chronic pancreatitis (Figs. 9, 10). Our results are It seems likely that this major difference in activation of NF-kB concordant with an investigation by Fukushima et al. (46) that signaling may help to explain the absence of LPS-related en- found Duox2 expression to be increased 13.6-fold in tissue adja- hancement of Duox2/DuoxA2 expression in the AsPC-1 line (Fig. cent to infiltrating pancreatic ductal carcinomas evaluated by 4D) versus that observed for BxPC-1 and CFPAC-1 cells. mRNA expression–array analysis. The requirement for upregulation of TLR4-related signaling in Finally, it is important to note that chronic pancreatic inflam- the control of Duox2 expression by IFN-g and LPS was sub- mation, in addition to being a premalignant condition, can result in stantiated by the RNA interference studies shown in Fig. 3; siR- inflammation-related pancreatic fibrosis, which can produce severe NAs targeting different sites on TLR4 decreased TLR4 expression clinical consequences (1, 2). Recent studies in model systems and decreased Duox2 and DuoxA2 expression at the RNA level, suggest that inflammation can be interrupted by treatment with as well as the expression of Duox protein, without altering the in- inhibitors of the Nox gene family, leading to the prevention of crease in activated Stat1 levels that we observed previously fol- many of the ROS-related morphologic consequences of chronic lowing exposure to IFN-g alone (24). These results are consistent inflammation in the pancreas (3). with prior studies demonstrating that LPS-induced reactive oxy- In summary, our experiments demonstrate that the proin- gen production in the THP-1 human monocyte line required flammatory stimuli IFN-g and LPS significantly enhance the TLR4-mediated signaling through the NF-kB pathway (44), as transcription of Duox2 and DuoxA2, leading to the production of well as that a direct interaction between TLR4 and the Nox4 substantial amounts of extracellular H2O2 by human pancreatic isoform is required to sustain LPS-mediated ROS generation and cancer cell lines, and that H2O2 generation may depend on the activation of NF-kB in HEK293 cells that express Nox4 (25). Our activation of both Stat1- and NF-kB–signaling pathways. Fur- Downloaded from finding that exposure of BxPC-3 cells to IFN-g and LPS leads to thermore, expression of Duox appears to be increased in human enhanced binding of NF-kB (p65) to the Duox2 promoter strongly pancreatic cancer models in vivo and in chronic pancreatitis in suggests that two concomitant mechanisms interact to produce man. In light of these data, support exists for the possibility that the marked increase in Duox2 expression that we observed (Fig. Duox2-related H2O2 production could provide an oxidative local 1). LPS alone enhances p65 binding to the Duox2 promoter; the milieu that enhances leukocyte recruitment and increases genetic

combination of LPS and IFN-g increases p65 binding further. In instability and that is sufficiently proangiogenic to be conducive http://www.jimmunol.org/ complementary fashion, IFN-g by itself appears to increase Duox2 to malignant transformation (9, 47, 48). Our ongoing studies are expression by increasing the binding of Stat1 to the Duox2 pro- focused on how an understanding of the pathophysiologic con- moter (24). We suggest that the combined transcriptional activa- sequences of increased Duox2 expression in pancreatic cancer tion produced by p65 and Stat1 is responsible for the dramatic could lead to novel methods to interrupt proinflammatory cycles increase in Duox2 expression and function observed in these of oxidative injury in the gastrointestinal tract. experiments. Cytokine-induced Duox2 expression in BxPC-3 cells also sig- Disclosures nificantly increased extracellular H O production in the presence 2 2 The authors have no financial conflicts of interest. by guest on September 26, 2021 or absence of stimulation by ionomycin (Fig. 6). The H2O2 levels produced were of sufficient magnitude to decrease the prolifera- tion of BxPC-3 cells by ∼50% (Fig. 6E); decreased tumor cell References growth was associated with a significant block in the G1 phase of 1. Farrow, B., and B. M. Evers. 2002. Inflammation and the development of pan- the cell cycle and markedly enhanced apoptosis (Fig. 7A, 7B). In creatic cancer. Surg. Oncol. 10: 153–169. 2. Greer, J. B., and D. C. Whitcomb. 2009. Inflammation and pancreatic cancer: an contrast, H2O2 production in the AsPC-1 line, which upregulates evidence-based review. Curr. Opin. Pharmacol. 9: 411–418. Duox2 following IFN-g treatment to a lesser degree than BxPC-3 3. Masamune, A., T. Watanabe, K. Kikuta, K. Satoh, and T. Shimosegawa. 2008. cells, was only modestly changed under these conditions (Fig. 6D) NADPH oxidase plays a crucial role in the activation of pancreatic stellate cells. Am. J. Physiol. Gastrointest. Liver Physiol. 294: G99–G108. and was not associated with any change in proliferation. Exposure 4. Yu, J. H., J. W. Lim, H. Kim, and K. H. Kim. 2005. NADPH oxidase mediates to IFN-g and LPS also produced DNA double-strand breaks (as interleukin-6 expression in cerulein-stimulated pancreatic acinar cells. Int. J. measured by an increase in phosphorylated gH2AX) in BxPC-3 Biochem. Cell Biol. 37: 1458–1469. 5. Guerra, C., M. Collado, C. Navas, A. J. Schuhmacher, I. Herna´ndez-Porras, cells that could be diminished by ROS scavengers (Fig. 7C). This M. Can˜amero, M. Rodriguez-Justo, M. Serrano, and M. Barbacid. 2011. degree of oxidant stress is consistent with a role for Duox2- Pancreatitis-induced inflammation contributes to pancreatic cancer by inhibiting oncogene-induced senescence. Cancer Cell 19: 728–739. mediated ROS in producing LPS-dependent injury to the extra- 6. Vaquero, E. C., M. Edderkaoui, S. J. Pandol, I. Gukovsky, and A. S. Gukovskaya. cellular matrix (30), as well as contributing to tumor cell genomic 2004. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis instability. These results also support recent studies suggesting that in pancreatic cancer cells. J. Biol. Chem. 279: 34643–34654. 7. Edderkaoui, M., P. Hong, E. C. Vaquero, J. K. Lee, L. Fischer, H. Friess, increased Duox2 expression in the intestinal epithelial cells of M. W. Buchler, M. M. Lerch, S. J. Pandol, and A. S. Gukovskaya. 2005. Ex- patients with Crohn’s disease and ulcerative colitis may have path- tracellular matrix stimulates reactive oxygen species production and increases ophysiologic significance (21). pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase. Am. J. Physiol. Gastrointest. Liver Physiol. 289: G1137–G1147. We recently demonstrated that Duox2 can be highly expressed 8. Mochizuki, T., S. Furuta, J. Mitsushita, W. H. Shang, M. Ito, Y. Yokoo, in human colorectal cancers in comparison with adjacent normal M. Yamaura, S. Ishizone, J. Nakayama, A. Konagai, et al. 2006. Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating colonic tissue (14). Other investigators (45) reported a 10-fold kinase 1 pathway in pancreatic cancer PANC-1 cells. Oncogene 25: 3699–3707. increase in Duox2 expression in premalignant adenomatous pol- 9. Chiera, F., E. Meccia, P. Degan, G. Aquilina, D. Pietraforte, M. Minetti, yps in the large intestine compared with adjacent normal tissue in D. Lambeth, and M. Bignami. 2008. Overexpression of human NOX1 complex induces genome instability in mammalian cells. Free Radic. Biol. Med. 44: 332– the same individuals, further supporting a role for Duox2 in ma- 342. lignant transformation. A role for Duox2 in both human pancreatic 10. Driessens, N., S. Versteyhe, C. Ghaddhab, A. Burniat, X. De Deken, J. Van cancer and chronic pancreatitis is supported by our in vivo ex- Sande, J. E. Dumont, F. Miot, and B. Corvilain. 2009. Hydrogen peroxide induces DNA single- and double-strand breaks in thyroid cells and is therefore periments demonstrating the rapid upregulation of Duox2 ex- a potential mutagen for this organ. Endocr. Relat. Cancer 16: 845–856. pression in two human tumor cell lines (BxPC-3 and AsPC-1) 11. Ostman, A., C. Hellberg, and F. D. Bo¨hmer. 2006. Protein-tyrosine phosphatases and cancer. Nat. Rev. Cancer 6: 307–320. grown as xenografts, but not for an IFN-g–unresponsive line 12. Szatrowski, T. P., and C. F. Nathan. 1991. Production of large amounts of hy- (MIA PaCa cells), as well as the increased staining for Duox in drogen peroxide by human tumor cells. Cancer Res. 51: 794–798. 14 INDUCTION OF DUOX2 BY IFN-g AND LPS IN PANCREATIC CANCER

13. Bedard, K., and K. H. Krause. 2007. The NOX family of ROS-generating 31. Ochi, A., A. H. Nguyen, A. S. Bedrosian, H. M. Mushlin, S. Zarbakhsh, NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87: 245–313. R. Barilla, C. P. Zambirinis, N. C. Fallon, A. Rehman, Y. Pylayeva-Gupta, et al. 14. Juhasz, A., Y. Ge, S. Markel, A. Chiu, L. Matsumoto, J. van Balgooy, K. Roy, 2012. MyD88 inhibition amplifies dendritic cell capacity to promote pancreatic and J. H. Doroshow. 2009. Expression of NADPH oxidase homologues and carcinogenesis via Th2 cells. J. Exp. Med. 209: 1671–1687. accessory genes in human cancer cell lines, tumours and adjacent normal tissues. 32. Grasberger, H., and S. Refetoff. 2006. Identification of the maturation factor for Free Radic. Res. 43: 523–532. dual oxidase. Evolution of an eukaryotic operon equivalent. J. Biol. Chem. 281: 15. Caillou, B., C. Dupuy, L. Lacroix, M. Nocera, M. Talbot, R. Ohayon, D. De`me, 18269–18272. J. M. Bidart, M. Schlumberger, and A. Virion. 2001. Expression of reduced 33. Bosisio, D., N. Polentarutti, M. Sironi, S. Bernasconi, K. Miyake, G. R. Webb, nicotinamide adenine dinucleotide phosphate oxidase (ThoX, LNOX, Duox) M. U. Martin, A. Mantovani, and M. Muzio. 2002. Stimulation of toll-like re- genes and proteins in human thyroid tissues. J. Clin. Endocrinol. Metab. 86: ceptor 4 expression in human mononuclear by interferon-gamma: 3351–3358. a molecular basis for priming and synergism with bacterial lipopolysaccharide. 16. Harper, R. W., C. Xu, J. P. Eiserich, Y. Chen, C. Y. Kao, P. Thai, H. Setiadi, and Blood 99: 3427–3431. R. Wu. 2005. Differential regulation of dual NADPH oxidases/peroxidases, 34. Abreu, M. T., E. T. Arnold, L. S. Thomas, R. Gonsky, Y. Zhou, B. Hu, and Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium. M. Arditi. 2002. TLR4 and MD-2 expression is regulated by immune-mediated FEBS Lett. 579: 4911–4917. signals in human intestinal epithelial cells. J. Biol. Chem. 277: 20431–20437. 17. El Hassani, R. A., N. Benfares, B. Caillou, M. Talbot, J. C. Sabourin, V. Belotte, 35. Quandt, K., K. Frech, H. Karas, E. Wingender, and T. Werner. 1995. MatInd and S. Morand, S. Gnidehou, D. Agnandji, R. Ohayon, et al. 2005. Dual oxidase2 is MatInspector: new fast and versatile tools for detection of consensus matches in expressed all along the digestive tract. Am. J. Physiol. Gastrointest. Liver nucleotide sequence data. Nucleic Acids Res. 23: 4878–4884. Physiol. 288: G933–G942. 36. Chen, Q. M., J. C. Bartholomew, J. Campisi, M. Acosta, J. D. Reagan, and 18. Allaoui, A., A. Botteaux, J. E. Dumont, C. Hoste, and X. De Deken. 2009. Dual B. N. Ames. 1998. Molecular analysis of H2O2-induced senescent-like growth oxidases and hydrogen peroxide in a complex dialogue between host mucosae arrest in normal human fibroblasts: p53 and Rb control G1 arrest but not cell and bacteria. Trends Mol. Med. 15: 571–579. replication. Biochem. J. 332: 43–50. 19. Geiszt, M., J. Witta, J. Baffi, K. Lekstrom, and T. L. Leto. 2003. Dual oxidases 37. Redon, C. E., A. J. Nakamura, Y. W. Zhang, J. J. Ji, W. M. Bonner, R. J. Kinders, represent novel hydrogen peroxide sources supporting mucosal surface host R. E. Parchment, J. H. Doroshow, and Y. Pommier. 2010. Histone gammaH2AX defense. FASEB J. 17: 1502–1504. and poly(ADP-ribose) as clinical pharmacodynamic biomarkers. Clin. Cancer 20. Gattas, M. V., R. Forteza, M. A. Fragoso, N. Fregien, P. Salas, M. Salathe, and Res. 16: 4532–4542. Downloaded from G. E. Conner. 2009. Oxidative epithelial host defense is regulated by infectious 38. Kolls, J. K. 2006. Oxidative stress in : a redox redux. J. Clin. Invest. 116: and inflammatory stimuli. Free Radic. Biol. Med. 47: 1450–1458. 860–863. 21.Lipinski,S.,A.Till,C.Sina,A.Arlt,H.Grasberger,S.Schreiber,and 39. Yu, M., J. Lam, B. Rada, T. L. Leto, and S. J. Levine. 2011. Double-stranded P. Rosenstiel. 2009. DUOX2-derived reactive oxygen species are effectors of RNA induces shedding of the 34-kDa soluble TNFR1 from human airway epi- NOD2-mediated antibacterial responses. J. Cell Sci. 122: 3522–3530. thelial cells via TLR3-TRIF-RIP1-dependent signaling: roles for dual oxidase 2- 22. Ha, E. M., K. A. Lee, Y. Y. Seo, S. H. Kim, J. H. Lim, B. H. Oh, J. Kim, and and caspase-dependent pathways. J. Immunol. 186: 1180–1188. W. J. Lee. 2009. Coordination of multiple dual oxidase-regulatory pathways in 40. Rada, B., and T. L. Leto. 2010. Characterization of hydrogen peroxide pro-

responses to commensal and infectious microbes in drosophila gut. Nat. duction by Duox in bronchial epithelial cells exposed to Pseudomonas aerugi- http://www.jimmunol.org/ Immunol. 10: 949–957. nosa. FEBS Lett. 584: 917–922. 23. Csillag, C., O. H. Nielsen, B. Vainer, J. Olsen, B. K. Dieckgraefe, J. Hendel, 41. Nagai, K., T. Betsuyaku, M. Suzuki, Y. Nasuhara, K. Kaga, S. Kondo, and I. Vind, C. Dupuy, F. C. Nielsen, and R. Borup. 2007. Expression of the genes M. Nishimura. 2008. and 2 expression in airway epithelium of dual oxidase 2, lipocalin 2 and regenerating islet-derived 1 alpha in Crohn’s smokers and patients with mild/moderate chronic obstructive pulmonary disease. disease. Scand. J. Gastroenterol. 42: 454–463. Antioxid. Redox Signal. 10: 705–714. 24. Wu, Y., S. Antony, A. Juhasz, J. Lu, Y. Ge, G. Jiang, K. Roy, and J. H. Doroshow. 42. Chen, R., A. B. Alvero, D. A. Silasi, K. D. Steffensen, and G. Mor. 2008. 2011. Up-regulation and sustained activation of Stat1 are essential for interferon- Cancers take their Toll—the function and regulation of Toll-like receptors in gamma (IFN-gamma)-induced dual oxidase 2 (Duox2) and dual oxidase A2 cancer cells. Oncogene 27: 225–233. (DuoxA2) expression in human pancreatic cancer cell lines. J. Biol. Chem. 286: 43. Gloire, G., S. Legrand-Poels, and J. Piette. 2006. NF-kappaB activation by re- 12245–12256. active oxygen species: fifteen years later. Biochem. Pharmacol. 72: 1493–1505. 25. Park, H. S., H. Y. Jung, E. Y. Park, J. Kim, W. J. Lee, and Y. S. Bae. 2004. 44. Ryan, K. A., M. F. Smith, Jr., M. K. Sanders, and P. B. Ernst. 2004. Reactive oxygen

Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is and nitrogen species differentially regulate Toll-like receptor 4-mediated activation by guest on September 26, 2021 essential for lipopolysaccharide-induced production of reactive oxygen species of NF-kappa B and interleukin-8 expression. Infect. Immun. 72: 2123–2130. and activation of NF-kappa B. J. Immunol. 173: 3589–3593. 45. Kita, H., Y. Hikichi, K. Hikami, K. Tsuneyama, Z. G. Cui, H. Osawa, 26. Ogier-Denis, E., S. B. Mkaddem, and A. Vandewalle. 2008. NOX enzymes and H. Ohnishi, H. Mutoh, H. Hoshino, C. L. Bowlus, et al. 2006. Differential gene Toll-like receptor signaling. Semin. Immunopathol. 30: 291–300. expression between flat adenoma and normal mucosa in the colon in a micro- 27. Testro, A. G., and K. Visvanathan. 2009. Toll-like receptors and their role in array analysis. J. Gastroenterol. 41: 1053–1063. gastrointestinal disease. J. Gastroenterol. Hepatol. 24: 943–954. 46. Fukushima, N., J. Koopmann, N. Sato, N. Prasad, R. Carvalho, S. D. Leach, 28. Zhang, X., C. Zhu, D. Wu, and X. Jiang. 2010. Possible role of toll-like receptor R. H. Hruban, and M. Goggins. 2005. Gene expression alterations in the non- 4 in acute pancreatitis. Pancreas 39: 819–824. neoplastic parenchyma adjacent to infiltrating pancreatic ductal adenocarcinoma. 29. Sharif, R., R. Dawra, K. Wasiluk, P. Phillips, V. Dudeja, E. Kurt-Jones, Mod. Pathol. 18: 779–787. R. Finberg, and A. Saluja. 2009. Impact of toll-like receptor 4 on the severity of 47. Niethammer, P., C. Grabher, A. T. Look, and T. J. Mitchison. 2009. A tissue- acute pancreatitis and pancreatitis-associated lung injury in mice. Gut 58: 813– scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. 819. Nature 459: 996–999. 30. Ikebe, M., Y. Kitaura, M. Nakamura, H. Tanaka, A. Yamasaki, S. Nagai, J. Wada, 48. Arbiser, J. L., J. Petros, R. Klafter, B. Govindajaran, E. R. McLaughlin, K. Yanai, K. Koga, N. Sato, et al. 2009. Lipopolysaccharide (LPS) increases the L. F. Brown, C. Cohen, M. Moses, S. Kilroy, R. S. Arnold, and J. D. Lambeth. invasive ability of pancreatic cancer cells through the TLR4/MyD88 signaling 2002. Reactive oxygen generated by Nox1 triggers the angiogenic switch. Proc. pathway. J. Surg. Oncol. 100: 725–731. Natl. Acad. Sci. USA 99: 715–720.