GroEL1, a Heat Shock Protein 60 of Chlamydia pneumoniae, Induces Lectin-Like Oxidized Low-Density Lipoprotein Receptor 1 Expression in Endothelial Cells and This information is current as Enhances Atherogenesis in of October 6, 2021. Hypercholesterolemic Rabbits Feng-Yen Lin, Yi-Wen Lin, Chun-Yao Huang, Yu-Jia Chang, Nai-Wen Tsao, Nen-Chung Chang, Keng-Liang Ou, Ta-Liang Chen, Chun-Ming Shih and Yung-Hsiang Chen Downloaded from J Immunol 2011; 186:4405-4414; Prepublished online 7 March 2011; doi: 10.4049/jimmunol.1003116 http://www.jimmunol.org/content/186/7/4405 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2011/03/07/jimmunol.100311 Material 6.DC1 References This article cites 47 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/186/7/4405.full#ref-list-1 by guest on October 6, 2021

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

GroEL1, a Heat Shock Protein 60 of Chlamydia pneumoniae, Induces Lectin-Like Oxidized Low-Density Lipoprotein Receptor 1 Expression in Endothelial Cells and Enhances Atherogenesis in Hypercholesterolemic Rabbits

Feng-Yen Lin,*,†,‡ Yi-Wen Lin,*,‡ Chun-Yao Huang,*,†,‡ Yu-Jia Chang,x Nai-Wen Tsao,{ Nen-Chung Chang,*,† Keng-Liang Ou,‡ Ta-Liang Chen,‖,#,1 Chun-Ming Shih,*,†,1 and Yung-Hsiang Chen**

Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) plays a major role in oxidized low-density lipoprotein-induced vascular inflammation. Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic patho- genesis, although its specific mechanism remains unknown. This study was conducted to investigate the mechanisms of LOX-1 Downloaded from expression in GroEL1 (a heat shock protein from C. pneumoniae)-administered human coronary artery endothelial cells (HCAECs) and atherogenesis in hypercholesterolemic rabbits. We demonstrated that in the hypercholesterolemic rabbit model, GroEL1 administration enhanced fatty streak and macrophage infiltration in atherosclerotic lesions, which may be mediated by elevated LOX-1 expression. In in vitro study using HCAECs, stimulation with GroEL1 increased TLR4 and LOX-1 expression. Increased LOX-1 expression was downregulated by Akt activation and PI3K-mediated endothelial NO synthase activation. PI3K http://www.jimmunol.org/ inhibitor and NO synthase inhibitor induced LOX-1 mRNA production, whereas the NO donor ameliorated the increasing effect of LOX-1 mRNA in GroEL1-stimulated HCAECs. LOX-1 expression was regulated by NADPH oxidase, which mediates reactive oxygen species production and intracellular MAPK signaling pathway in GroEL1-stimulated HCAECs. Treatment with poly- ethylene-glycol–conjugated superoxide dismutase, apocynin, or diphenylene iodonium significantly decreased GroEL1-induced LOX-1 expression, as did the knockdown of Rac1 gene expression by RNA interference. In conclusion, the GroEL1 protein may induce LOX-1 expression in endothelial cells and atherogenesis in hypercholesterolemic rabbits. The elevated level of LOX-1 in vitro may be mediated by the PI3K–Akt signaling pathway, endothelial NO synthase activation, NADPH oxidase-mediated reactive oxygen species production, and MAPK activation in GroEL1-stimulated HCAECs. The GroEL1 protein of C. pneumoniae may contribute to vascular inflammation and cardiovascular disorders. The Journal of Immunology, 2011, 186: 4405–4414. by guest on October 6, 2021

hlamydia pneumoniae is a Gram-negative bacterium that onstrated that C. pneumoniae accelerates the formation of com- has a biphasic elementary body (EB) and reticulate body plex atherosclerosis in ApoE3-Leiden mice (4) and can induce C life cycle. There is extensive evidence indicating that increased uptake of low-density lipoprotein (LDL) in macroph- C. pneumoniae may play a key role in the development of ath- ages (5). Although C. pneumoniae can induce LDL oxidation erosclerosis and coronary artery disease (1). Clinical evidence within the neointima (6) and may potentially increase the de- has shown that early fatty streaks present accumulation of C. velopment of hypercholesterolemia-mediated atherosclerosis, there pneumoniae, and seropositivity is associated with increasing vascu- is no direct evidence of C. pneumoniae infection preceding oxi- lar intima-media thickness (2). Inoculation of C. pneumoniae in dized LDL (oxLDL)-mediated atherogenesis. animals may induce the formation of atherosclerotic lesions in hy- During the infectious process of C. pneumoniae in target cells, percholesterolemic conditions (3). However, another study dem- EBs require both attachment to and phagocytosis by host cells

*Division of Cardiology, Taipei Medical University Hospital, Taipei 110, Taiwan; Medicine, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei 110, Taiwan {Division of Cardiovascular Surgery, Taipei Medical University Hospital, Taipei 110, (C.-M.S.). E-mail addresses: [email protected] (Y.-H.C.) and cmshih53@ ‖ Taiwan; Department of Anesthesiology, Taipei Medical University Hospital, Taipei tmu.edu.tw (C.-M.S.) 110, Taiwan; †Department of Internal Medicine, Taipei Medical University, Taipei The online version of this article contains supplemental material. 110, Taiwan; #Department of Anesthesiology, School of Medicine, College of Med- icine, Taipei Medical University, Taipei 110, Taiwan; xGraduate Institute of Clinical Abbreviations used in this article: ALT, alanine aminotransferase; AST, aspartate Medicine, Taipei Medical University, Taipei 110, Taiwan; ‡Research Center For Bio- aminotransferase; BUN, blood urea nitrogen; BW, body weight; CRP, C-reactive medical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, protein; DiI, 1,19-dioctadecyl-3,3,39,39-tetramethyl-indocarbocyanine perchlorate; DPI, Taiwan; and **Graduate Institute of Integrated Medicine, China Medical University, diphenylene iodonium; EB, elementary body; eNOS, endothelial NO synthase; ESR, Taichung 40402, Taiwan erythrocyte sedimentation rate; HC, high cholesterol; HCAEC, human coronary artery g endothelial cell; hTLR, human TLR; LDL, low-density lipoprotein; L-NAME, L-N - 1T.-L.C. and C.-M.S. contributed equally to this work. nitro-L-arginine methyl ester; LOX-1, lectin-like oxidized LDL receptor 1; oxLDL, Received for publication September 17, 2010. Accepted for publication January 20, oxidized LDL; PAK, p21-activated kinase; PBD, p21 binding domain; ROS, reactive 2011. oxygen species; SAPK, stress-activated protein kinase; siRNA, small interfering RNA; SNOC, S-nitrosocysteine; SR-B1, scavenger receptor B1; SREC, scavenger This work was supported by National Science Council, Taiwan Grants NSC 97-2314- receptor expressed by endothelial cell. B-038-035-MY2 and 99-2314-B-038-030-MY2. Address correspondence and reprint requests to Dr. Yung-Hsiang Chen and Dr. Chun- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 Ming Shih, Graduate Institute of Integrated Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan (Y.-H.C.) and Department of Internal www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003116 4406 C. PNEUMONIAE GroEL1 INDUCES LOX-1

(7). Heat shock protein 60 of C. pneumoniae (GroEL) is also were pelleted by centrifugation for 10 min at 8000 rpm, and rGroEL was expressed on the surface of EBs, and the proteins could be fallen extracted under native conditions according to the protocol of GST Gene off from the EBs. Reports have demonstrated that the EB surface- Fusion System manufacturer’s instructions (GE Healthcare Amersham Biosciences). Finally, rGroEL protein was purified by elution buffer con- associated GroEL1 is the major binding adhesion protein and taining 50 mM Tris-HCl and 10 mM reduced glutathione (pH 8.0). The plays an important role in the pathogenesis of infectious diseases quantity of rGroEL protein was measured using the Bio-Rad protein assay (8). In clinical practice, the GroEL1 protein indeed causes several (Bio-Rad, Hercules, CA). The fusion protein was detected by SDS gel complicated diseases, including respiratory tract diseases and vas- electrophoresis, and identified by immunoblotting with a GST Ab (GE Healthcare Amersham Biosciences). Endotoxin levels in the rGroEL pro- cular diseases (9). Additionally, the GroEL1 protein may initiate tein were measured using a Limulus amebocyte lysate kit from Cambrex. the secretion of IL-6 and TNF-a in dendritic cells, and stimulate LPS levels were below 1 pg/ml. production of IL-1b, -6, and -8 and proliferation in vascular cells Measurement of GroEL1 protein cytotoxicity and activity and mononuclear cells by triggering intracellular signaling path- way activation within eukaryotic cells (10–12). Cell cytotoxicity of rGroEL protein was analyzed by the MTT assay. One crucial event for C. pneumonia-induced atherosclerosis is HCAECs were grown in 96-well plates and incubated with various con- centrations (1–500 ng/ml) of rGroEL for 24 h. Subsequently, 0.5 mg/ml believed to be oxLDL accumulation, which results in the formation MTT was added to each well, and incubation was continued at 37˚C for of foam cells. C. pneumonia infection enhances lectin-like oxidized an additional 4 h. DMSO was added to each well, and the absorbance was LDL receptor 1 (LOX-1), but not scavenger receptor expressed by recorded at 530 nm using a DIAS Microplate Reader (Dynex Technolo- gies). The activity of rGroEL was measured by ELISA. Human monocytic endothelial cell (SREC) expression (13), which accelerates ath- 6 erogenesis in the presence of hypercholesterolemia. We hypothe- cell line THP-1 cells were seeded in 24-well plates at a density of 10 cells/ ml/well, and these were then treated with various concentrations of GroEL sized that C. pneumoniae GroEL1 protein may increase LOX-1

protein (1–100 ng/ml) for 1 or 3 h. The culture medium was collected to Downloaded from expression in the endothelium, which mediates oxLDL uptake and quantify the levels of TNF-a using the DuoSet ELISA development kits the development of serious atherosclerosis. Therefore, we also (R&D Systems), and the absorbance was recorded using a DIAS Micro- examined whether GroEL1 increased neointimal hyperplasia and plate Reader (Dynex Technologies). LOX-1 expression in hypercholesterolemic rabbits. Furthermore, Animal experiment we explored the cellular events and underlying mechanisms in- All animals were treated according to protocols approved by the In- volved in GroEL1-induced LOX-1 expression using human coro- stitutional Animal Care Committee of the Taipei Medical University nary artery endothelial cells (HCAECs) in vitro. (Taiwan). Experimental procedures and animal care conformed to the Guide http://www.jimmunol.org/ for the Care and Use of Laboratory Animals published by the United States Materials and Methods National Institutes of Health (Publication 85-23, revised 1996). Fifteen adult male New Zealand White rabbits (2.5–3 kg) were used. After 1 wk on Production and purification of C. pneumonia elementary a commercial rabbit chow diet (Scientific Diet Services, Essex, U.K.) at bodies 60 g/kg/d with water ad libitum, nine animals were placed on a 2% high- cholesterol (HC) diet (Purina Mills, St. Louis, MO) and six animals were C. pneumonia (TWARTW-183) were cultured on Hela 229 cells, and 100 ml placed on a normal diet. The animals were divided into five groups, as inoculum from 270˚C storage was used in HEPES/sucrose/citrate dilution. follows: group 1 was the control; group 2 received the HC diet; group 3 Plates were centrifuged 1 h at 70 3 g to enhance cellular attachment. The received the HC diet and i.v. injections of GroEL1 (2 mg/kg body weight) supernatant was aspirated and discarded, and chlamydial growth media was

through the ear vein at the end of weeks 2, 3, and 4; group 4 received the by guest on October 6, 2021 added. Plates were incubated at 36˚C with CO for 3 d, and then infected 2 HC diet and i.v. injections of GroEL1 (4 mg/kg body weight) at the end of cells were harvested. The harvested cells were sonicated for 15 s, then weeks 2, 3, and 4; and group 5 received the normal chow diet and GroEL1 centrifugated at 700 3 g for 15 min, and then pelleted at 18,000 3 g at 4˚C (4 mg/kg body weight) at the end of weeks 2, 3, and 4. The animals were for 30 min. The pellet was resuspended in HEPES-sucrose-cation solution. sacrificed and the abdominal aortas were then harvested, gently dissected The 3–5 ml suspension was layered over a 13 ml HEPES-sucrose-cation free of adherent tissues, rinsed with ice-cold PBS, immersion fixed with adding 7 ml Renografin and centrifuged 18,000 3 g at 4˚C for 30 min. The 4% buffered paraformaldehyde, paraffin embedded, and then cross-sec- final suspension containing EBs was to the original volume in 0.01 M PBS. tioned for morphometry (H&E staining) and immunohistochemistry. The Construction of C. pneumoniae GroEL expression vectors thoracic aortas were also collected and stained with Sudan IV solution for visualization of the fatty streak areas. The genomic DNA of C. pneumonia was extraction from EBs using EasyPure Genomic DNA mini kit (Bioman Scientific, Taipei, Taiwan). Measurement of erythrocyte sedimentation rate and serum Segment containing the open reading frame of the GroEL was originally C-reactive protein PCR amplified by using 100 ng C. pneumonia genomic DNA as template, 0.2 mM dNTPs, 1 mM each of gene-specific primers, and 1 U PFU DNA Erythrocyte sedimentation rate (ESR) and serum C-reactive protein (CRP) polymerase (Promega, Madison, WI) with the following program: 1 cycle levels were analyzed to evaluate GroEL1-induced systemic inflammatory of 95˚C for 5 min, 38 cycles of 95˚C for 45 s, 68˚C for 45 s, and 72˚C for 2 responses. Arterial blood was collected from the ear artery into tubes min; 1 cycle of 68˚C for 45 s and 72˚C for 10 min; and a final incubation containing sodium citrate. ESR is used primarily to detect occult processes at 72˚C for 10 min with 1 U Taq DNA polymerase. The following gene- and monitor inflammatory conditions. Rabbit ESR was measured by the specific primers were used in the PCR reaction: GroEL Pr-forward, 59- Westergren method. CRP is regarded as an acute-phase reactant in the serum CGAATTCTTAAGGAGAACAACGATGGCAG-39 (forward primers con- in a wide variety of diseases. Rabbit serum was separated from whole blood, tained an EcoRI site) and GroEL Pr-reverse, 59-ACGGCCGGTAGTCCA- and CRP was then measured in triplicate with a commercial ELISA kit TTCCTGCGCTTGG C-39 (reverse primers contained an EagI site). The (Immunology Consultants Laboratory). amplified GroEL cDNA fragment was then cloned into pCR2.1-TOPO vec- Biochemical measurements tor (Invitrogen, Carlsbad, CA), and subsequently cloned in-frame into the EcoRI and NotI sites of pGEX-5X-1 expression vector (GE Healthcare Blood samples for biochemical measurements were collected from each Amersham Biosciences) for expression in Escherichia coli. animal before and at 2 and 5 wk of the experiment. Samples were separated by centrifugation, and the serum was stored at 280˚C until analysis. Serum Purification of GroEL recombinant protein total cholesterol and triglyceride were measured using Merck assay kits BL21 cells were transformed with pGEX-5X-1-GroEL expression vector (Darmstadt, Germany). Serum blood urea nitrogen (BUN), creatinine, al- and GroEL recombinant proteins were purified. Briefly, the BL21 cells anine aminotransferase (ALT), and aspartate aminotransferase (AST) were containing pGEX-5X-1-GroEL plasmid were grown overnight at 37˚C in also measured using a SPOTCHEMTM automatic dry chemistry system 2 ml Luria-Bertani medium supplemented with 100 mg/ml ampicillin. (SP-4410; Arkray, Shanghai, Japan). Then 1.25 ml overnight culture was transferred into 100 ml Luria-Bertani/ Sudan IV staining of arteries for fatty streaks ampicillin medium and grown at 37˚C to an A600 of 0.6–0.8 (∼2 h). Fusion protein expression was then induced by adding isopropyl b-D-thi- The fixed thoracic aortas were immersed in 70% ethanol for 30 min and then ogalactoside to a final concentration of 1 mM at 30˚C for 6 h. Bacteria in the 1% Sudan IV/70% ethanol solution at room temperature for 2 h. The The Journal of Immunology 4407 samples were then dipped in 70% ethanol for 3 min and rinsed with running porated, according to the manufacturer’s instruction manual. The cells tap water. Sudanophilic areas were measured quantitatively by computer- were seeded into six-well plates immediately after transfection or, for assisted planimetry, and the extent of the lesions was expressed as a pro- further experiments, after 48 h. Silencer-validated siRNA (negative control portion of the total surface area (surface area of lesions/total surface area of siRNA; Ambion Catalog 4635) was used for knockdown validation. the thoracic aorta). NADPH oxidase activity assay Cell culture NADPH oxidase activity was determined with superoxide-dependent m HCAECs were purchased from Cascade Biologics (Portland, OR). Human lucigenin chemiluminescence, as previously described (18). The 40 g m monocytic THP-1 cells were purchased from American Type Culture membrane protein extraction and 5 M dark-adapted lucigenin were added m Collection (Manassas, VA). Cell cultures and passages were performed to a 96-well luminometer plate and adjusted to a final volume of 250 l m according to the manufacturer’s instructions. HCAECs were used at pas- with oxidase assay buffer before 100 M NADPH was added. Relative sages 3–8. Purity of the HCAEC cultures was verified by immunostaining light units were read with a luminometer (Dynatech ML2250; Dynatech with a mAb directed against smooth muscle-specific a-actin (R&D Sys- Laboratories). Light emission was recorded every minute for 15 min and tems, Minneapolis, MN). was expressed as mean relative light units/min. Pull-down assay for Rac1 activity Immunohistochemical and immunofluorescent staining Rac1 activation was measured using a GST-(p21-activated kinase)-p21 Immunohistochemical and immunofluorescent staining were performed on binding domain (GST-[PAK]-PBD) fusion protein, which binds to acti- serial 5-mm–thick paraffin-embedded sections of rabbit abdominal aortas vated Rac1. HCAECs were lysed in lysis buffer (25 mM HEPES, 150 mM and coverslip-grown HCAECs using anti–LOX-1, anti-SREC, scavenger NaCl, 1% Igepal CA-630, 10% glycerol, 10 mM MgCl , 1 mM EDTA, 10 receptor B1 (SR-B1), or anti–RAM-11 Abs. DAPI was used to identify the 2 mg/ml leupeptin, 10 mg/ml aprotinin). The supernatant was collected and nucleus. The slides were observed with microscopy or confocal micros- incubated with GST-(PAK)-PBD fusion protein. The protein–bead com- copy. Downloaded from plexes were then recovered by centrifugation and washed. Following the 9 9 9 last wash, the protein–bead complexes were resuspended in SDS reducing Uptake of 1,1 -dioctadecyl-3,3,3 ,3 -tetramethyl- sample buffer and resolved by 12% SDS-PAGE. The proteins were indocarbocyanine perchlorate–LDL by HCAECs transferred to polyvinylidene difluoride membrane, and the membrane was incubated with mouse anti-Rac1 Ab (Upstate Biotechnology) and HRP- Human LDL (d:1.019–1.063 g/ml) was isolated by sequential ultracentri- conjugated secondary Ab. Activated Rac1 was then detected using an ECL fugation of fasting plasma samples from healthy adult males (14). The detection kit. native LDL was oxidized, as described by Steinbrecher et al. (15, 16). The oxLDL was labeled with 1,19-dioctadecyl-3,3,39,39-tetramethyl-indocarbocya- Statistical analyses http://www.jimmunol.org/ nine perchlorate (DiI), as described previously (17). To examine cellular up- take of oxLDL, HCAECs were seated on culture slides and incubated for 4 h Values are expressed as means 6 SEM. Statistical evaluation was per- in cultured medium containing 80 mg/ml DiI-labeled oxLDL. At the end of formed using Student’s t test and one- or two-way ANOVA, followed by the treatment, cells were washed with PBS,mountedoncoverslips,andex- Dunnett’s test. A p value ,0.05 was considered significant. amined with confocal microscopy. Western blot analysis Results a Membrane fractions and total cell lysates were extracted from HCAECs. High concentrations of GroEL1 induce THP-1 cell TNF- Proteins were separated by SDS-PAGE and transferred to polyvinylidene production and HCAEC cytotoxicity difluoride membrane. The membranes were probed with mouse anti–LOX-1 by guest on October 6, 2021 (Santa Cruz Biotechnology), anti-SREC (Santa Cruz Biotechnology), anti– Treatment of HCAECs with 1, 10, 25, 50, 100, or 250 ng/ml SR-B1 (Santa Cruz Biotechnology), anti-endothelial NO synthase ([eNOS], GroEL1 protein for 24 h did not result in cell viability (Supple- Millipore). anti–phospho-eNOS (Millopore), anti-Akt (Anaspec), anti–phos- mental Fig. 1A). In contrast, a high concentration (500 ng/ml) of pho-Akt (Merck, Darmstadt, Geremany), goat anti-TLR4 (R&D Systems), GroEL1 protein may cause a significant reduction in cell viability. goat anti-TLR2 Ab (R&D Systems), rabbit anti-p38, rabbit anti–phospho- Treatment of THP-1 cells with 100 ng/ml GroEL1 for 1 h and p38, rabbit anti–stress-activated protein kinase (SAPK)/JNK, rabbit anti– a phospho-SAPK/JNK, rabbit anti-p44/p42 MAPK, or mouse anti–phospho- 1–100 ng/ml GroEL1 for 3 h may induce TNF- production (Sup- p44/p42 MAPK Ab (all MAPK Abs were purchased from Cell Signaling plemental Fig. 1B). The results indicated that rGroEL1 has bio- Technology). The proteins were visualized with an ECL detection kit activity and results in cell cytotoxicity at high concentrations. (Amersham Biosciences). Mouse anti–a-actin (Labvision/NeoMarkers) and rabbit anti-Gas (Santa Cruz Biotechnology) Abs were used as loading Biochemical measurements for rabbits controls. During the experimental period, weight gain and final weight did Quantitative real-time PCR not differ significantly between the groups of animals (data not shown). As shown in Table I, serum AST, ALT, BUN, and creati- Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA), nine levels also showed no significant difference between groups. according to the manufacturer’s instructions. LOX-1 and SREC mRNA expression were determined using quantitative real-time PCR. The level of Serum total cholesterol levels were increased after 2-wk HC diet. LOX-1 and SREC mRNA expression was determined in arbitrary units by At the end of the fifth experimental week, the HC diet may have the comparison with an external DNA standard, which was amplified with the elevated total cholesterol level. Compared with the control group, LOX-1 and SREC primers. PCR primers used for the amplification of GroEL1 protein treatment did not increase the serum total cho- LOX-1 and GAPDH mRNAs were as follows: LOX-1 forward primer, 59- AAGGACCAGCCTGATGAGAA-39 and reverse primer, 59-GCTGAGA- lesterol level. Additionally, serum triglyceride levels did not in- TCTGTCCCTCCAG-39; SREC forward primer, 59-CCTCAGCTCCCAC- crease significantly in any group during the experimental period. AAGCTAC-39 and reverse primer, 59-GAACCTCATCTGCCTCTCCA-39; SR-B1 forward primer, 59-CTGTGGGTGAGATCATGTGG-39 and reverse GroEL1 protein induces inflammatory responses in rabbits primer, 59-GCCAGAAGTCAACCTTGCTC-39; and GAPDH forward pri- ESR and serum CRP levels were analyzed to monitor GroEL1- mer, 59-TGCCCCCTCTGCTGATGCC-39 and reverse primer, 59-CCTCCG- ACGCCTGCTTCACCAC-39. induced systemic inflammation (Table II). In the control and HC diet groups, ESR and CRP levels did not change during the ex- Knockdown gene expression with interference RNA periment. However, ESR increased in the GroEL1 groups at weeks 4 and 5. CRP levels increased at week 3 and continued to increase Intracellular TLR2 and TLR4 expression was knocked down by transfection with interference RNA (small interfering RNA [siRNA]). Cells (106) were throughout the experimental period. During the experimental pe- trypsinized and resuspended in 100 ml Nucleofector solution (Amaxa riod, heart rate, rectal body temperature, and respiratory rate of the Biosystems), and 30 nM TLR2 or TLR4 siRNA (Ambion) was electro- rabbits were monitored. This result suggests that GroEL1 protein 4408 C. PNEUMONIAE GroEL1 INDUCES LOX-1

Table I. Plasma biochemical characteristics (n = 6) in experimental rabbits

HC Diet and GroEL1 Treatment GroEL1 Weeks Control HC Diet 4 mg/kg BW 2 mg/kg BW 4 mg/kg BW BUN (mg/dl) 0 19.32 6 5.32 20.34 6 4.89 23.44 6 6.78 19.23 6 8.90 21.09 6 5.66 2 16.43 6 3.45 21.02 6 6.43 21.90 6 6.43 21.55 6 5.78 20.45 6 7.34 5 21.66 6 6.88 19.22 6 4.56 19.39 6 3.67 20.18 6 5.34 18.99 6 4.32 Creatinine (mg/dl) 0 1.38 6 0.21 1.78 6 0.22 1.55 6 0.11 1.39 6 0.21 1.37 6 0.06 2 1.23 6 0.25 1.54 6 0.48 1.58 6 0.10 1.63 6 0.09 1.09 6 0.11 5 1.65 6 0.37 1.48 6 0.12 1.74 6 0.04 1.74 6 0.05 1.93 6 0.02 AST (IU/l) 0 15.33 6 4.21 17.32 6 7.87 18.71 6 2.43 16.32 6 7.21 17.92 6 4.53 2 16.94 6 7.64 15.82 6 4.89 20.82 6 6.88 16.99 6 3.69 17.00 6 3.79 5 19.92 6 5.09 19.03 6 3.55 16.90 6 4.76 20.02 6 5.89 21.90 6 5.87 ALT (IU/l) 0 55.82 6 9.43 48.99 6 10.32 48.81 6 7.43 46.72 6 7.43 47.99 6 10.92 2 51.90 6 11.32 61.23 6 12.93 47.70 6 8.64 54.90 6 4.68 51.23 6 5.74 5 47.92 6 6.90 58.92 6 6.99 39.90 6 5.92 43.76 6 8.37 46.25 6 8.66 Total cholesterol (mg/dl) 0 56.83 6 7.43 65.02 6 7.44 55.73 6 7.15 56.44 6 5.39 59.90 6 5.87 2 59.45 6 6.45 1554.21 6 192.22a 52.12 6 7.42 1632.32 6 178.43a 1521.87 6 190.21a 5 49.45 6 8.44 2312.12 6 198.34a 53.87 6 4.77 2473.22 6 160.30a 1982.32 6 166.33a Triglyceride (mg/dl) 0 40.54 6 7.43 50.21 6 8.32 45.43 6 5.76 45.90 6 4.33 46.32 6 7.32 2 47.98 6 6.44 47.53 6 7.38 48.96 6 5.34 48.96 6 6.90 51.34 6 6.44

5 43.01 6 8.12 61.09 6 9.02 50.83 6 8.12 59.99 6 7.21 58.20 6 7.12 Downloaded from Values are mean 6 SD. ap , 0.05 compared with 0 wk at the same group. injection may keep rabbits in a nonsepsis state and induce chronic infiltrated macrophages showed that fewer macrophages infiltrated combining acute systemic inflammation. into the vessel walls in the control, HC diet, and 4 mg/kg BW GroEL1 groups compared with the HC diet plus GroEL1 groups http://www.jimmunol.org/ GroEL1 enhances atherosclerotic lesion formation in HC (Fig. 1C). These results demonstrate that administration of GroEL1 diet-fed rabbits protein significantly increased macrophage infiltration and athero- Representative photographs of the fatty streaks of thoracic aortas sclerotic plaque formation in HC diet-fed rabbits. stained with Sudan IV from the five groups are shown in Fig. 1A. There were no atherosclerotic lesions in the aortas of control LOX-1 expression in GroEL1-administered rabbits rabbits. Sudanophilic atherosclerotic lesions in the HC diet group Immunohistochemical staining was performed using Abs against were slightly increasing under en face observation. A significant LOX-1, SREC, and SR-B1 on sections of the abdominal aortas (Fig. area of the aortic intimal surface from the HC diet plus 2 mg/kg 1D). Compared with sections from the control group, GroEL1 by guest on October 6, 2021 body weight (BW) GroEL1 group and the HC diet plus 4 mg/kg administration slightly enhanced LOX-1 expression in the endo- BW GroEL1 group was covered with fatty streaks. Furthermore, thelium of the HC diet group, whereas strong LOX-1 staining was use of 4 mg/kg BW GroEL1 induced more serious fatty streak seen on the luminal surface of the 4 mg/kg BW GroEL1 group and formation than use of 2 mg/kg BW GroEL1 in rabbits fed an HC in the neointima of the HC diet plus 4 mg/kg BW GroEL1 group. diet. In contrast, administration of 4 mg/kg BW GroEL1 protein In contrast, neither HC diet nor GroEL1 administration induced did not increase atherosclerotic lesion formation in rabbits fed a SREC and SR-B1 expression in the abdominal aorta in rabbits. normal chow diet. These results demonstrate that GroEL1 administration increased Fig. 1B shows that rabbits in the control, HC diet, and 4 mg/kg LOX-1 expression and significantly severe atherosclerotic lesion BW GroEL1 groups did not have thickened intima or athero- formation in the aorta. sclerotic lesion formation in the abdominal aortas. Abdominal aortas had slight lesion formation in the HC diet plus 2 mg/kg BW GroEL1 induces LOX-1 expression and enhances DiI-oxLDL GroEL1 group and marked lesion formation in the HC diet plus uptake in HCAECs 4 mg/kg BW GroEL1 group compared with these areas in the A DiI-oxLDL uptake assay was performed to investigate whether control group. Staining with anti–RAM-11 Ab for identification of oxLDL uptake is enhanced in GroEL1-stimulated endothelial cells.

Table II. Inflammatory characteristics (n = 6) in experimental rabbit

HC Diet and GroEL1 Treatment GroEL1 Weeks Control HC Diet 4 mg/kg BW 2 mg/kg BW 4 mg/kg BW ESR (mm/2 h) 0 2.2 6 1.2 2.3 6 0.9 2.0 6 0.5 1.4 6 0.3 1.7 6 1.0 3 2.4 6 1.4 1.9 6 0.4 2.0 6 0.6 2.1 6 0.7 2.3 6 0.6 4 1.9 6 1.6 2.1 6 0.6 13.8 6 1.6a,b 11.1 6 0.7a,b 17.8 6 0.9a,b 5 2.6 6 0.5 2.4 6 1.3 19.83 6 6.1a,b 18.9 6 5.2a,b 21.8 6 7.1a,b CRP (mg/dl) 0 0.88 6 0.03 0.83 6 0.12 0.91 6 0.74 1.17 6 0.53 1.23 6 0.69 3 1.32 6 0.48 1.03 6 0.42 18.05 6 0.78a,b 13.49 6 0.63a,b 17.29 6 0.98a,b 4 2.32 6 1.08 1.53 6 0.52 11.05 6 0.89a,b 10.99 6 0.11a,b 11.29 6 0.48a,b 5 1.36 6 0.32 1.90 6 0.14 19.83 6 1.29a,b 11.90 6 0.91a,b 16.12 6 2.79a,b Values are mean 6 SD. ap , 0.05 compared with control group at the same time point. bp , 0.05 compared with 0 wk at the same group. The Journal of Immunology 4409 Downloaded from http://www.jimmunol.org/ by guest on October 6, 2021

FIGURE 1. GroEL1 protein induces atherosclerotic lesion formation in HC diet-fed rabbits. A, Representative photographs of atherosclerotic lesions (fatty streak) of aortas stained with Sudan IV. The graph indicates the extent of atherosclerotic lesions in aortas from the five experimental groups. Results are expressed as mean 6 SEM. *p , 0.05. B, Histopathological features of cross-sections of abdominal aortas were stained using H&E. The lumen is uppermost in all sections, and the internal elastic laminae are indicated with arrows. C and D, Immunohistochemistry to assess infiltrated monocytic cells, LOX-1, SREC, and SR-B1 in rabbit abdominal aortas. Corresponding hematoxylin staining was used for nucleus identification.

Confocal microscopy demonstrated that treatment with GroEL1 petition assay, a nonspecific IgG2a isotype Ab was substituted protein for 24 h significantly increased DiI-oxLDL uptake in for the TLR4-specific Ab; however, the nonspecific Ab and the HCAECs (Fig. 2A). Real-time PCR demonstrated that GroEL1 silencer-validated siRNA did not affect GroEL1-induced LOX-1 treatment for 12 h may induce LOX-1 mRNA expression, but not mRNA production (data not shown). In contrast, Fig. 3C shows SREC or SR-B1 mRNA expression (Fig. 2B). Immunofluores- that mouse anti-hTLR2 Ab and TLR2 siRNA did not block LOX-1 cence also showed that treatment with GroEL1 protein for 24 h mRNA production in GroEL1-stimulated HCAECs, suggesting induces LOX-1 protein expression in HCAECs (Fig. 2C). LOX-1, the GroEL1-induced LOX-1 expression in HCAECs is mediated SREC, and SR-B1 were originally identified on the membrane of by TLR4. vascular endothelial cells; Western blotting showed that GroEL1 Akt signaling pathway and eNOS activity may be involved in protein increases intracellular LOX-1 production and elevates mem- GroEL1-induced LOX-1 expression brane LOX-1 expression in HCAECs (Fig. 2D). We next investigated the effects of GroEL1 protein on Akt kinase TLR4 mediates LOX-1 expression in GroEL1-stimulated signals and eNOS expression in HCAECs. After 6 h of incuba- HCAECs tion, Akt phosphorylation was significantly decreased in GroEL1 C. pneumonia GroEL1 may induce innate immune responses in protein-cultured HCAECs (Fig. 4A). Furthermore, eNOS phos- vitro via TLR2 and TLR4 (19). As shown in Fig. 3A, GroEL1 phorylation at Ser1177 was significantly decreased both in GroEL1 protein significantly induced TLR4 expression, but not TLR2 protein-stimulated and LY294002 (a PI3K inhibitor)-treated expression. Treatment with 100 ng/ml GroEL1 for 12 h signifi- HCAECs compared with control conditions (Fig. 4B). The PI3K– cantly induced LOX-1 mRNA expression (Fig. 3B,3C). Addition Akt signaling pathway did not involve eNOS production in GroEL1- of a mouse anti-human TLR (hTLR)4 Ab (10 mg/ml) and sub- stimulated HCAECs (LY294002 treatment did not decrease eNOS sequent incubation for 30 min or transfection with 25 nM TLR4 production; Fig. 4B); the potential roles of MAPK-related mech- siRNA prior to GroEL1 treatment significantly reduced LOX-1 anisms were also examined. Pretreatment with SB203580 (p38 mRNA production in HCAECs. As a negative control in the com- MAPK inhibitor), SP600125 (a JNK/SAPK inhibitor), or PD98059 4410 C. PNEUMONIAE GroEL1 INDUCES LOX-1 Downloaded from http://www.jimmunol.org/ FIGURE 2. GroEL1 protein induces LOX-1 expression and enhances oxLDL uptake in HCAECs. A, Intracellular DiI-oxLDL was observed using confocal microscopy. B, Expression of LOX-1, SREC, and SR-B1 mRNA was measured by real-time PCR. Data represent the results of three independent experiments (mean 6 SEM). *p , 0.05 compared with the unstimulated group. C, Expression of LOX-1, SREC, and SR-B1 in HCAECs was detected by immunofluorescence and observed by confocal microscopy. DAPI was used to stain the HCAEC nuclei. D, Membrane and total LOX-1, SREC, or SR-B1 protein levels were analyzed with Western blotting. b-actin and Gas were used as loading controls. The values represent quantification of the protein expression (fold of control) in HCAECs.

(an MEK1 antagonist) for 1 h did not ameliorate the inhibitory fusion protein pull-down assay. HCAECs were treated with by guest on October 6, 2021 effects of GroEL1 protein on eNOS phosphorylation (Fig. 4C). 25–100 ng/ml GroEL1 protein for 120 min. Total cell lysates were Interestingly, GroEL1-stimulated decreases in eNOS production extracted and measured for Rac1 activity. GroEL1 treatment were reduced by SB203580, but not by SP600125 or PD98059. rapidly induced Rac1 activation in a dosage-dependent manner This result probably indicated that GroEL1 protein influences (Fig. 5B). To explore whether NADPH-mediated reactive oxygen eNOS production mediating by p38 MAPK. The potential roles of species (ROS) are involved in GroEL1-induced LOX-1 expres- PI3K/Akt and eNOS in LOX-1 expression in GroEL1-stimulated sion, HCAECs were pretreated with various antioxidants or trans- HCAECs were then examined. Coincubation with NO donor fected with Rac1 siRNA prior to stimulation with GroEL1 protein. S-nitrosocysteine (SNOC) significantly ameliorated the increases As shown in Fig. 5C, GroEL1 or H2O2 significantly induced in LOX-1 mRNA (Fig. 4D) and LOX-1 protein (Fig. 4E)on LOX-1 mRNA expression, which was significantly blocked by GroEL1-stimulated HCAECs. In contrast, incubation with NO pretreatment with polyethylene-glycol–conjugated superoxide dis- g synthase inhibitor L-N -nitro-L-arginine methyl ester (L-NAME) or mutase (a permeable antioxidant enzyme), apocynin (a specific PI3K inhibitor LY294002 significantly enhanced LOX-1 expres- NADPH oxidase inhibitor), or DPI for 1 h. GroEL1-induced LOX-1 sion. These data indicate that GroEL1 protein may upregulate mRNA expression was completely blocked by Rac1 when a RNA LOX-1 expression in endothelial cells by modulating PI3K/Akt-, silencing technique was used, but not by the negative control eNOS-, and p38 MAPK-related mechanisms. siRNA. These results suggest that GroEL1-induced LOX-1 ex- pression is mediated by an oxidative stress-related mechanism GroEL1-induced LOX-1 expression is mediated by Rac1 and and that the NADPH oxidase subunit Rac1 plays a critical role in NADPH oxidase activation and ROS generation LOX-1 regulation. To determine whether GroEL1 protein induces NADPH oxidase activation in HCAECs, HCAECs were treated with 100 ng/ml Intracellular MAPK signaling in GroEL1-induced LOX-1 GroEL1 protein for 0 (d), 60 (s), 90 (▼), or 120 (4) min, and expression the membrane fraction was then assayed for NADPH oxidase GroEL1 protein markedly induced the phosphorylation of MAPKs, activity (Fig. 5A). GroEL1 protein treatment resulted in a time- including p38 MAPK, ERK1/2, and SAPK/JNK (Fig. 5D), after dependent increase in enzyme activity. Diphenylene iodonium exposure to 25–100 ng/ml GroEL1 protein for 30 min. DPI sig- (DPI; 100 mM) was used to determine NADPH oxidase activity nificantly decreased SAPK/JNK activation in GroEL1-stimulated (Fig. 5A), which is associated with Rac1 activation insofar as it HCAECs, but did not influence p38 MAPK or ERK1/2 activation has been reported to play important roles in NADPH oxidase (Fig. 5D), suggesting that NADPH-oxidase–derived ROS are in- activation (20). To further examine whether GroEL1-induced volved in SAPK–JNK signaling pathway activation. Real-time NADPH oxidase activation is accompanied by increases in Rac1 PCR demonstrated that GroEL1-induced LOX-1 mRNA expres- activation, we measured activated Rac1 with a GST-(PAK)-PBD sion was reduced by SB203580 (a p38 MAPK inhibitor), SP600125 The Journal of Immunology 4411

on serological markers. Although these results do not conclusively rule out a role of C. pneumoniae in the pathogenesis of athero- sclerosis, they definitively do not support the use of antibiotics in the secondary prevention of coronary heart disease. Our data showed that C. pneumoniae GroEL1 protein alone did not induce fatty streak formation, but did enhance the formation of athero- sclerotic lesions in HC diet-fed rabbits, suggesting the potential role of C. pneumoniae heat shock proteins in atherosclerosis in- duction in the presence of hypercholesterolemia. This may pro- vide a potential rationale for the dramatic failure of clinical trials using antibiotics for atherosclerosis. Studies have shown associations between atherosclerosis and different pathogens, and early investigations showed the presence of infectious pathogens in the whole arterial vessel tree (27). It is known that pathogens such as C. pneumoniae can induce macro- phage foam cell formation (28), an effect that might be increased if an individual has been infected by multiple pathogens (5). TLR/ MyD88 and liver X receptor signaling pathways reciprocally control C. pneumoniae-induced acceleration of atherosclerosis (29). It has been hypothesized that the infectious pathogen contains Downloaded from proteins that are homologous to parts of the host proteins, resulting in an immune response called infection-induced molecular mim- icry (30). It has recently been shown that C. pneumoniae heat shock protein 60 localizes in human atheroma and regulates mac- rophage TNF-a and matrix metalloproteinase expression (31); in fact, this heat shock protein 60 can activate human vascular en- http://www.jimmunol.org/ dothelium, smooth muscle cells, and macrophages (10), and can stimulate cellular LDL oxidation in vitro (6). Our data show that administration of GroEL1 protein significantly increased macro- phage infiltration in atherosclerotic plaques in HC diet-fed rabbits, FIGURE 3. GroEL1-induced LOX-1 expression in HCAECs is medi- supporting the results that genital Chlamydia infections are asso- ated by TLR4. A, TLR4 and TLR2 protein expression was analyzed. B, ciated with significant induction of the chemokines and chemokine HCAECs were pretreated with anti-hTLR4 Ab or transfected with TLR4 receptors that are involved in the recruitment of immune cells into siRNA prior to GroEL1 treatment. LOX-1 mRNA expression was analyzed infection sites (32). by guest on October 6, 2021 by real-time PCR. C, HCAECs were pretreated with anti-hTLR2 Ab or The hypercholesterolemic with GroEL1 administration rabbit transfected with TLR2 siRNA prior to GroEL1 treatment. LOX-1 mRNA model was used in the current study. Rabbits were injected with 2 expression was analyzed by real-time PCR. Data represent the results of or 4 mg/kg body weight GroEL1, which represents a GroEL1 level three independent experiments (mean 6 SEM). *p , 0.05. of 50 or 100 ng/ml plasma, respectively. The combined results of ESR and serum CRP measurements are a useful indicator of in- flammation. The rectal body temperature and respiratory rate of the (a SAPK/JNK inhibitor), or SB203580 plus SP600125, but not by rabbits were kept in the normal range (the normal range of rectal PD98059 (a MEK1 antagonist) (Fig. 5E). These results suggest body temperature, 38–40˚C and respiratory rate, 30–60 breaths/ that SAPK/JNK and p38 MAPK play more significant roles than min) after GroEL1 was administered. Elevated CRP and ESR, ERK1/2 in the GroEL1-mediated LOX-1 mRNA expression tran- but the maintenance of normal vital signs, indicated that the dose scriptional regulatory signaling pathway. of GroEL1 was sufficient to produce inflammation in the animals, which remained in a nonseptic state. Indeed, we decided the dose Discussion of GroEL1 in the in vivo study with 25–100 ng/ml was consistent In the current study, to our knowledge, we demonstrated for the first with the pathophysiological state of hypercholesterolemic and time that C. pneumoniae GroEL1 protein may enhance LOX-1 GroEL1-administrated rabbit study in vivo. Previous findings have expression in endothelium and mediate oxLDL uptake and ath- demonstrated that a repertoire of LOX-1 is associated with ath- erosclerosis progression. In fact, GroEL1 increased neointimal erosclerotic lesions. Although atherosclerotic plaque lipids can be hyperplasia and LOX-1 expression in hypercholesterolemic rab- found in macrophages and foam cells, and LOX-1 is not only bits. Furthermore, our in vitro findings suggest that GroEL1 in- expressed by endothelial cells, but also by smooth muscle cells teracts with TLR4 and plays critical roles in oxLDL uptake of endo- and macrophages, we therefore cannot exclude that macrophages/ thelial cells, which results from elevated LOX-1 expression and smooth muscle cells via the GroEL1/LOX-1 route play some roles is mediated by NADPH oxidase activation, PI3K/Akt-mediated during atherogenesis. Further studies are required to clarify the eNOS activation, and the MAPK signaling pathways. interaction between GroEL1 and LOX-1 expression in smooth muscles and monocytes. C. pneumonia infection and atherogenesis The role of C. pneumoniae infection in the pathogenesis of ath- C. pneumonia and LOX-1 expression erosclerosis remains controversial (21–24). Meta-analyses of ran- The histopathological features of C. pneumoniae-induced aortic domized clinical trials of antibiotic therapy for secondary pre- lesions closely resemble early changes produced by a diet enriched vention of coronary heart disease did not show any benefit (25, with low amounts (0.15%) of cholesterol in rabbits, which resulted 26). There was also no effect of possible C. pneumoniae infection in serum cholesterol levels (4.1 mM) similar to what is recom- 4412 C. PNEUMONIAE GroEL1 INDUCES LOX-1

FIGURE 4. GroEL1 protein upregu- lates LOX-1 expression in HCAECs by modulating PI3K/Akt-, eNOS-, and p38 MAPK-related mechanisms. A, HCAECs were treated for 6 h with 25–100 mg/ ml GroEL1 protein, and Akt activation (phosphorylation) was analyzed by West- ern blot analysis. B, HCAECs were treated for 8 h with 25–100 mg/ml GroEL1 pro- tein or 5 mM LY294002 (LY), and eNOS phosphorylation and production were analyzed. C, HCAECs were pretreated with 10 mM SB203580 (SB), SP600125 (SP), or PD98059 (PD) for 1 h prior to stimulation with GroEL1 protein for 12 h. D, LOX-1 mRNA expression was ana-

lyzed after 12 h of culture in control or Downloaded from GroEL1 protein in the absence or pres- ence of SNOC (100 mM)-containing, L- NAME (10 mM)-containing, or LY294002 (10 mM)-containing medium. Data repre- sent the results of three independent ex- periments (mean 6 SEM). *p , 0.05. E,

LOX-1 protein expression was observed http://www.jimmunol.org/ using confocal microscopy after 24 h of culture in control or GroEL1 protein in the absence or presence of SNOC-containing, L-NAME–containing, or LY294002-con- taining medium. by guest on October 6, 2021

mended for humans (33). C. pneumoniae infection was also found In the current study, exogenous addition of rGroEL1 induced to accelerate the development of atherosclerosis in rabbits fed a oxLDL uptake and LOX-1 expression in endothelial cells. TLR4 cholesterol-enriched diet (34). Our data were inconsistent with was the mediator in GroEL1-induced LOX-1 expression. These previous studies in which C. pneumoniae was detected in the results suggest that the GroEL1-induced expression of LOX-1 in atherosclerotic aorta in cholesterol-fed or apolipoprotein E– HCAECs is mediated by TLR4. This result is inconsistent with the deficient mice, but did not induce vascular changes by itself (35); observation that the mitogenic effect of C. pneumoniae on vascular in mice with LDL receptor and apolipoprotein E deficiencies, smooth muscle cells could be mimicked by exogenous Chlamydia C. pneumoniae was found to exacerbate hypercholesterolemia- heat shock protein 60 via a TLR4-mediated signaling pathway induced atherosclerosis (36, 37). (12). Several groups have reported that TLR4 expression is up- LOX-1 is a scavenger receptor that functions as the primary regulated by bacterial LPS and certain cytokines in endothelial oxLDL receptor in endothelial cells and is implicated in oxLDL- cells (40); extracellular heat shock protein 60 may still act as a induced endothelial dysfunction and atherosclerosis. Recently, signal transducer to stimulate cells via upregulated TLR4. much emphasis has been placed on oxLDL as a major mediator of endothelial activation and/or dysfunction. Specific oxLDL recep- Regulation of LOX-1 tors including LOX-1 and scavenger receptors have been described Induced endothelial expression of LOX-1 upon various pathologic in endothelial cells, macrophages, and smooth muscle cells. These stimuli would provide a molecular link for incorporation of oxLDL receptors facilitate uptake of oxLDL and subsequent endothelial into cells, resulting in cellular activation, dysfunction, and injury activation, transformation of macrophages to foam cells, and (41). Because oxLDL has been shown to generate ROS, tethering smooth muscle cell proliferation (38, 39). In the current study, we oxLDL via LOX-1 would focus oxidant stress on cellular targets, demonstrated that GroEL1 upregulates LOX-1 expression in resulting in changes in gene expression and cellular phenotype HCAECs. Because LOX-1 is the main receptor for oxLDL and and, in some cases, induction of endothelial dysfunction. Recent may play an important role in the pathogenesis of atherosclerosis, studies showed that oxLDL binding to LOX-1 in endothelial cells identification and regulation of LOX-1 and understanding its signal induces generation of superoxide anion (42). As DPI, a selective transduction pathways might improve our insight of the patho- inhibitorofNADPHoxidase,drastically reduced intracellular genesis of atherosclerosis and provide a selective treatment ap- superoxide concentration, oxLDL-induced superoxide generation proach. LOX-1 might be a potential and promising target for the might be related to increased NADPH oxidase activity. The in- development of novel anti-atherosclerotic drugs. creased intracellular superoxide anion accelerates NO inactiva- The Journal of Immunology 4413 Downloaded from http://www.jimmunol.org/

FIGURE 5. NADPH-oxidase–mediated ROS generation and MAPK signaling pathways are involved in LOX-1 expression. A, NADPH oxidase activity was measured with a superoxide-dependent lucigenin chemiluminescent assay. B, HCAECs were treated with GroEL1 protein. Cytosolic Rac1 activity was measured using a pull-down assay. C, HCAECs were pretreated with polyethylene-glycol–conjugated superoxide dismutase, apocynin (APO), or DPI, or

were transfected with 25 nM Rac1 siRNA, followed by GroEL1 protein stimulation for 12 h. LOX-1 mRNA expression levels were analyzed by quantitative by guest on October 6, 2021 real-time PCR. H2O2 was used as the positive control. Silencer-validated siRNA (NC siRNA) was used for knockdown validation. D, HCAECs were treated with GroEL1 in the presence or absence of 100 mM DPI before the cell lysates were extracted. Phosphorylation of p38 MAPK, ERK1/2, and SAPK/JNK was analyzed by Western blotting. E, HCAECs were pretreated with 10 mM SB203580, PD98059, or SP600125 for 1 h before treatment with 100 ng/ml GroEL1 protein. LOX-1 mRNA levels were evaluated by quantitative real-time PCR. Data represent the results of three independent experiments (mean 6 SEM; *p , 0.05). tion (43). The presence of oxLDL decreases intracellular NO con- kinase activation involves oxidative stress and that MAPKs act centrations in basal, bradykinin-stimulated, and thrombin-stim- in this signaling cascade as intermediate molecules that carry sig- ulated conditions. In addition, in HCAECs, oxLDL decreases nals from NADPH oxidase to endothelial LOX-1. PKB/Akt phosphorylation and downregulates eNOS activity (44). In this study, we had noted the preferred target of the various Our data also demonstrated that GroEL1 induced LOX-1 expres- inhibitors/antagonists that were used in cellular analyses. We had sion in the Akt–eNOS-related pathway, which suggests that the performed the pilot study for the possible cytotoxicity amount of Akt–eNOS-related pathway plays an important role in GroEL1- these various kinase and oxidase inhibitors with MTT assay. No induced LOX-1 activation. apparent cytotoxicity was found for the dosages that were used in Production of oxLDL promotes free radical generation and the study. Actually, the various kinase and oxidase inhibitors that causes lipid peroxidation in endothelial cells (45). LOX-1 acti- are used in this study can often have off-target effects in various vation is associated with intracellular free radical generation, cell lines and types. In the future, we should have more direct evi- which has a positive effect on LOX-1 expression (42). Regulation dence to verify the influence of GroEL1 on LOX-1 expression in of LOX-1 gene expression is redox sensitive (46); therefore, ROS HCAECs. generated by GroEL1 in vascular cells may be key intermediates In conclusion, our results demonstrated that the C. pneumoniae in the regulation of LOX-1 gene expression. Evidence linking GroEL1 protein may induce LOX-1 expression in endothelial oxidative stress with MAPK activation in vascular cells addi- cells, which mediates fatty streak formation in atherogenesis. tionally supports a role of these kinases in the control of LOX-1 The elevated level of LOX-1 may be mediated by the PI3K–Akt expression (47). In line with these hypotheses, we found that anti- signaling pathway, eNOS activation, NADPH oxidase-mediated oxidants and NADPH oxidase/MAPK inhibitors reduced GroEL1- ROS production, and MAPK activation in GroEL1-stimulated induced LOX-1 levels, thus implicating ROS and kinases as sig- HCAECs. The C. pneumoniae GroEL1 protein may contribute naling molecules in this effect. Our findings that antioxidants to vascular inflammation and cardiovascular disorders. Our work suppressed GroEL1-induced NADPH oxidase/MAPK activation identifying LOX-1 as a target gene for GroEL1 provides a basis and that NADPH oxidase inhibition abolished GroEL1-induced for further investigation of LOX-1 modulation as a therapeutic MAPK activation support the hypothesis that GroEL1-induced strategy for atherogenesis in C. pneumoniae infection. 4414 C. PNEUMONIAE GroEL1 INDUCES LOX-1

Acknowledgments 25. Wells, B. J., A. G. Mainous, III, and L. M. Dickerson. 2004. Antibiotics for the secondary prevention of ischemic heart disease: a meta-analysis of randomized We thank Tze-Liang Yang and Min-Yu Lo for excellent technical assistance. controlled trials. Arch. Intern. Med. 164: 2156–2161. 26. Illoh, K. O., O. C. Illoh, H. B. Feseha, and J. M. Hallenbeck. 2005. Antibiotics for vascular diseases: a meta-analysis of randomized controlled trials. Athero- Disclosures sclerosis 179: 403–412. The authors have no financial conflicts of interest. 27. Saikku, P., M. Leinonen, L. Tenkanen, E. Linnanma¨ki, M. R. Ekman, V. Manninen, M. Ma¨ntta¨ri, M. H. Frick, and J. K. Huttunen. 1992. Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. Ann. Intern. Med. 116: 273–278. References 28. Chen, S., R. Sorrentino, K. Shimada, Y. Bulut, T. M. Doherty, T. R. Crother, and 1. Xu, Q. 2003. Infections, heat shock proteins, and atherosclerosis. Curr. Opin. M. Arditi. 2008. Chlamydia pneumoniae-induced foam cell formation requires Cardiol. 18: 245–252. MyD88-dependent and -independent signaling and is reciprocally modulated by 2. Volanen, I., M. J. Ja¨rvisalo, R. Vainionpa¨a¨, M. Arffman, K. Kallio, S. Angle´, liver X receptor activation. J. Immunol. 181: 7186–7193. T. Ro¨nnemaa, J. Viikari, J. Marniemi, O. T. Raitakari, and O. Simell. 2006. 29. Naiki, Y., R. Sorrentino, M. H. Wong, K. S. Michelsen, K. Shimada, S. Chen, Increased aortic intima-media thickness in 11-year-old healthy children with A. Yilmaz, A. Slepenkin, N. W. Schro¨der, T. R. Crother, et al. 2008. TLR/ persistent Chlamydia pneumoniae seropositivity. Arterioscler. Thromb. Vasc. MyD88 and liver X receptor alpha signaling pathways reciprocally control Biol. 26: 649–655. Chlamydia pneumoniae-induced acceleration of atherosclerosis. J. Immunol. 3. Blessing, E., L. A. Campbell, M. E. Rosenfeld, N. Chough, and C. C. Kuo. 2001. 181: 7176–7185. Chlamydia pneumoniae infection accelerates hyperlipidemia induced athero- 30. Wick, G., H. Perschinka, and Q. Xu. 1999. Autoimmunity and atherosclerosis. sclerotic lesion development in C57BL/6J mice. Atherosclerosis 158: 13–17. Am. Heart J. 138: S444–S449. 4. Ezzahiri, R., H. J. Nelissen-Vrancken, H. A. Kurvers, F. R. Stassen, I. Vliegen, 31. Kol, A., G. K. Sukhova, A. H. Lichtman, and P. Libby. 1998. Chlamydial heat G. E. Grauls, M. M. van Pul, P. J. Kitslaar, and C. A. Bruggeman. 2002. Chla- shock protein 60 localizes in human atheroma and regulates macrophage tumor mydophila pneumoniae (Chlamydia pneumoniae) accelerates the formation of necrosis factor-alpha and matrix metalloproteinase expression. Circulation 98: complex atherosclerotic lesions in Apo E3-Leiden mice. Cardiovasc. Res. 56: 300–307. 32. Belay, T., F. O. Eko, G. A. Ananaba, S. Bowers, T. Moore, D. Lyn, and 269–276. Downloaded from 5. Kalayoglu, M. V., and G. I. Byrne. 1998. Induction of macrophage foam cell J. U. Igietseme. 2002. Chemokine and chemokine receptor dynamics during formation by Chlamydia pneumoniae. J. Infect. Dis. 177: 725–729. genital chlamydial infection. Infect. Immun. 70: 844–850. 6. Kalayoglu, M. V., B. Hoerneman, D. LaVerda, S. G. Morrison, R. P. Morrison, 33. Fong, I. W., B. Chiu, E. Viira, D. Jang, and J. B. Mahony. 1999. De novo in- and G. I. Byrne. 1999. Cellular oxidation of low-density lipoprotein by Chla- duction of atherosclerosis by Chlamydia pneumoniae in a rabbit model. Infect. mydia pneumoniae. J. Infect. Dis. 180: 780–790. Immun. 67: 6048–6055. 7. Hybiske,K.,andR.S.Stephens.2007.Mechanismsofhostcellexitbytheintracellular 34. Muhlestein, J. B., J. L. Anderson, E. H. Hammond, L. Zhao, S. Trehan, bacteriumChlamydia.Proc.Natl.Acad.Sci.USA104:11430–11435. E. P. Schwobe, and J. F. Carlquist. 1998. Infection with Chlamydia pneumoniae 8. Zu¨gel, U., and S. H. Kaufmann. 1999. Role of heat shock proteins in protection accelerates the development of atherosclerosis and treatment with azithromycin from and pathogenesis of infectious diseases. Clin. Microbiol. Rev. 12: 19–39. prevents it in a rabbit model. Circulation 97: 633–636. http://www.jimmunol.org/ 9. Borel, N., J. T. Summersgill, S. Mukhopadhyay, R. D. Miller, J. A. Ramirez, and 35. Jackson, D. J., J. P. Rakwar, B. Chohan, K. Mandaliya, J. J. Bwayo, J. O. Ndinya- A. Pospischil. 2008. Evidence for persistent Chlamydia pneumoniae infection of Achola, N. J. Nagelkerke, J. K. Kreiss, and S. Moses. 1997. Urethral infection in human coronary atheromas. Atherosclerosis 199: 154–161. a workplace population of East African men: evaluation of strategies for 10. Kol, A., T. Bourcier, A. H. Lichtman, and P. Libby. 1999. Chlamydial and human screening and management. J. Infect. Dis. 175: 833–838. heat shock protein 60s activate human vascular endothelium, smooth muscle 36. Hu, H., G. N. Pierce, and G. Zhong. 1999. The atherogenic effects of chlamydia cells, and macrophages. J. Clin. Invest. 103: 571–577. are dependent on serum cholesterol and specific to Chlamydia pneumoniae. 11. Kru¨ll, M., A. C. Klucken, F. N. Wuppermann, O. Fuhrmann, C. Magerl, J. Clin. Invest. 103: 747–753. J. Seybold, S. Hippenstiel, J. H. Hegemann, C. A. Jantos, and N. Suttorp. 1999. 37. Moazed, T. C., L. A. Campbell, M. E. Rosenfeld, J. T. Grayston, and C. C. Kuo. Signal transduction pathways activated in endothelial cells following infection 1999. Chlamydia pneumoniae infection accelerates the progression of athero- with Chlamydia pneumoniae. J. Immunol. 162: 4834–4841. sclerosis in apolipoprotein E-deficient mice. J. Infect. Dis. 180: 238–241. 38. Tsukamoto, K., M. Kinoshita, K. Kojima, Y. Mikuni, M. Kudo, M. Mori,

12. Sasu, S., D. LaVerda, N. Qureshi, D. T. Golenbock, and D. Beasley. 2001. by guest on October 6, 2021 Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate pro- M. Fujita, E. Horie, N. Shimazu, and T. Teramoto. 2002. Synergically increased liferation of human vascular smooth muscle cells via Toll-like receptor 4 and expression of CD36, CLA-1 and CD68, but not of SR-A and LOX-1, with the progression to foam cells from macrophages. J. Atheroscler. Thromb. 9: 57–64. p44/p42 mitogen-activated protein kinase activation. Circ. Res. 89: 244–250. 39. Boullier, A., D. A. Bird, M. K. Chang, E. A. Dennis, P. Friedman, K. Gillotre- 13. Yoshida, T., N. Koide, I. Mori, H. Ito, and T. Yokochi. 2006. Chlamydia pneu- Taylor, S. Horkko, W. Palinski, O. Quehenberger, P. Shaw, et al. 2001. Scavenger moniae infection enhances lectin-like oxidized low-density lipoprotein receptor receptors, oxidized LDL, and atherosclerosis. Ann. N. Y. Acad. Sci. 947: 214– (LOX-1) expression on human endothelial cells. FEMS Microbiol. Lett. 260: 222, discussion 222–213. 17–22. 40. Faure, E., L. Thomas, H. Xu, A. Medvedev, O. Equils, and M. Arditi. 2001. 14. Winocour, P. H., P. N. Durrington, D. Bhatnagar, M. Ishola, S. Arrol, and Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and M. Mackness. 1992. Abnormalities of VLDL, IDL, and LDL characterize Toll-like receptor 4 expression in human endothelial cells: role of NF-kappa B Arterioscler. Thromb. insulin-dependent diabetes mellitus. 12: 920–928. activation. J. Immunol. 166: 2018–2024. 15. Steinbrecher, U. P., S. Parthasarathy, D. S. Leake, J. L. Witztum, and 41. Sakurai, K., and T. Sawamura. 2003. Stress and vascular responses: endothelial D. Steinberg. 1984. Modification of low density lipoprotein by endothelial cells dysfunction via lectin-like oxidized low-density lipoprotein receptor-1: close involves lipid peroxidation and degradation of low density lipoprotein phos- relationships with oxidative stress. J. Pharmacol. Sci. 91: 182–186. pholipids. Proc. Natl. Acad. Sci. USA 81: 3883–3887. 42. Cominacini, L., A. F. Pasini, U. Garbin, A. Davoli, M. L. Tosetti, 16. Hatch, F. T. 1968. Practical methods for plasma lipoprotein analysis. Adv. Lipid M. Campagnola, A. Rigoni, A. M. Pastorino, V. Lo Cascio, and T. Sawamura. Res. 6: 1–68. 2000. Oxidized low density lipoprotein (ox-LDL) binding to ox-LDL receptor-1 17. Oriol, A., J. M. Ribera, J. Arnal, F. Milla, M. Batlle, and E. Feliu. 1993. Sac- in endothelial cells induces the activation of NF-kappaB through an increased charomyces cerevisiae septicemia in a patient with myelodysplastic syndrome. production of intracellular reactive oxygen species. J. Biol. Chem. 275: 12633– Am. J. Hematol. 43: 325–326. 12638. 18. Smith, K. R., L. R. Klei, and A. Barchowsky. 2001. Arsenite stimulates plasma 43. Cominacini, L., A. Rigoni, A. F. Pasini, U. Garbin, A. Davoli, M. Campagnola, membrane NADPH oxidase in vascular endothelial cells. Am. J. Physiol. Lung A. M. Pastorino, V. Lo Cascio, and T. Sawamura. 2001. The binding of oxidized Cell. Mol. Physiol. 280: L442–L449. low density lipoprotein (ox-LDL) to ox-LDL receptor-1 reduces the intracellular 19. Costa, C. P., C. J. Kirschning, D. Busch, S. Du¨rr, L. Jennen, U. Heinzmann, concentration of nitric oxide in endothelial cells through an increased production S. Prebeck, H. Wagner, and T. Miethke. 2002. Role of chlamydial heat shock of superoxide. J. Biol. Chem. 276: 13750–13755. protein 60 in the stimulation of innate immune cells by Chlamydia pneumoniae. 44. Li, D. Y., H. J. Chen, and J. L. Mehta. 2001. Statins inhibit oxidized-LDL- Eur. J. Immunol. 32: 2460–2470. mediated LOX-1 expression, uptake of oxidized-LDL and reduction in PKB 20. Yokoyama, M., N. Inoue, and S. Kawashima. 2000. Role of the vascular NADH/ phosphorylation. Cardiovasc. Res. 52: 130–135. NADPH oxidase system in atherosclerosis. Ann. N. Y. Acad. Sci. 902: 241–247, 45. Vergnani, L., S. Hatrik, F. Ricci, A. Passaro, N. Manzoli, G. Zuliani, discussion 247–248. V. Brovkovych, R. Fellin, and T. Malinski. 2000. Effect of native and oxidized 21. Belland, R. J., S. P. Ouellette, J. Gieffers, and G. I. Byrne. 2004. Chlamydia low-density lipoprotein on endothelial nitric oxide and superoxide production: pneumoniae and atherosclerosis. Cell. Microbiol. 6: 117–127. key role of L-arginine availability. Circulation 101: 1261–1266. 22. Grayston, J. T. 2000. Background and current knowledge of Chlamydia pneu- 46. Nagase, M., K. Ando, T. Nagase, S. Kaname, T. Sawamura, and T. Fujita. 2001. moniae and atherosclerosis. J. Infect. Dis. 181(Suppl. 3): S402–S410. Redox-sensitive regulation of lox-1 gene expression in vascular endothelium. 23. Ieven, M. M., and V.Y.Hoymans. 2005. Involvement of Chlamydia pneumoniae in Biochem. Biophys. Res. Commun. 281: 720–725. atherosclerosis: more evidence for lack of evidence. J. Clin. Microbiol. 43: 19–24. 47. Li, L., T. Sawamura, and G. Renier. 2004. Glucose enhances human macrophage 24. Kalayoglu, M. V., P. Libby, and G. I. Byrne. 2002. Chlamydia pneumoniae as an LOX-1 expression: role for LOX-1 in glucose-induced macrophage foam cell emerging risk factor in cardiovascular disease. JAMA 288: 2724–2731. formation. Circ. Res. 94: 892–901.