CCAAT/Enhancer-Binding Protein δ Is a Critical Mediator of Lipopolysaccharide-Induced Acute Lung Injury
2012; Elsevier BV; Volume: 182; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2012.10.013
ISSN1525-2191
AutoresChunguang Yan, Peter F. Johnson, Huifang Tang, Yan Ye, Min Wu, Hongwei Gao,
Tópico(s)Respiratory Support and Mechanisms
ResumoAlthough inflammation plays a central role in the pathogenesis of acute lung injury, the molecular mechanisms underlying inflammatory responses in acute lung injury are poorly understood, and therapeutic options remain limited. CCAAT/enhancer-binding proteins, C/EBPβ and C/EBPδ, are expressed in the lung and have been implicated in the regulation of inflammatory mediators. However, their functions in lung pathobiological characteristics are not well characterized. Herein, we show that C/EBPβ and C/EBPδ are activated in mouse lung after intrapulmonary deposition of lipopolysaccharide (LPS). Mice carrying a targeted deletion of the C/EBPδ gene displayed significant attenuation of the lung permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), and neutrophils in bronchial alveolar lavage fluids compared with wild-type mice. These phenotypes were consistent with morphological evaluation of lung, which showed reduced inflammatory cell influx and minimal intra-alveolar hemorrhage. Moreover, mutant mice expressed considerably less tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 in bronchial alveolar lavage fluids in LPS-injured lung compared with wild-type mice. In contrast, C/EBPβ deficiency had no effect on LPS-induced lung injury. By using small-interfering RNA–mediated knockdown for C/EBPδ, we demonstrate, for the first time to our knowledge, that C/EBPδ plays a critical role for the tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 production in LPS-stimulated alveolar macrophages. These findings demonstrate that C/EBPδ, but not C/EBPβ, plays an important role in LPS-induced lung inflammatory responses and injury. Although inflammation plays a central role in the pathogenesis of acute lung injury, the molecular mechanisms underlying inflammatory responses in acute lung injury are poorly understood, and therapeutic options remain limited. CCAAT/enhancer-binding proteins, C/EBPβ and C/EBPδ, are expressed in the lung and have been implicated in the regulation of inflammatory mediators. However, their functions in lung pathobiological characteristics are not well characterized. Herein, we show that C/EBPβ and C/EBPδ are activated in mouse lung after intrapulmonary deposition of lipopolysaccharide (LPS). Mice carrying a targeted deletion of the C/EBPδ gene displayed significant attenuation of the lung permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), and neutrophils in bronchial alveolar lavage fluids compared with wild-type mice. These phenotypes were consistent with morphological evaluation of lung, which showed reduced inflammatory cell influx and minimal intra-alveolar hemorrhage. Moreover, mutant mice expressed considerably less tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 in bronchial alveolar lavage fluids in LPS-injured lung compared with wild-type mice. In contrast, C/EBPβ deficiency had no effect on LPS-induced lung injury. By using small-interfering RNA–mediated knockdown for C/EBPδ, we demonstrate, for the first time to our knowledge, that C/EBPδ plays a critical role for the tumor necrosis factor-α, IL-6, and macrophage inflammatory protein-2 production in LPS-stimulated alveolar macrophages. These findings demonstrate that C/EBPδ, but not C/EBPβ, plays an important role in LPS-induced lung inflammatory responses and injury. Acute lung injury affects approximately 190,000 patients annually in the United States.1Tsushima K. King L.S. Aggarwal N.R. De Gorordo A. D'Alessio F.R. 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Low-level vancomycin resistance in Staphylococcus aureus: an Australian perspective.Eur J Clin Microbiol Infect Dis. 2005; 24: 100-108Crossref PubMed Scopus (17) Google Scholar C/EBPβ and C/EBPδ are structurally similar in their DNA-binding and dimerization domains, but differ in their transactivation domains, implying that they may have unique functions in response to different stimuli. Both C/EBPβ and C/EBPδ are expressed in the lung.20Fong Y. Shen K.H. Chen L.J. Cheng J.T. Changes of CCAAT enhancer-binding proteins (CEBPs) in the lung of streptozotocin-induced diabetic rats.Horm Metab Res. 2011; 43: 261-267Crossref PubMed Scopus (3) Google Scholar, 21Browder W. Ha T. Chuanfu L. Kalbfleisch J.H. Ferguson Jr., D.A. Williams D.L. Early activation of pulmonary nuclear factor kappaB and nuclear factor interleukin-6 in polymicrobial sepsis.J Trauma. 1999; 46: 590-596Crossref PubMed Scopus (38) Google Scholar, 22Miglino N. Roth M. Lardinois D. Sadowski C. Tamm M. Borger P. 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Differential expression of three C/EBP isoforms in multiple tissues during the acute phase response.J Biol Chem. 1992; 267: 5021-5024Abstract Full Text PDF PubMed Google Scholar By using a gene expression profiling approach, a recent study identified C/EBPδ as a potential candidate regulator of endotoxin-induced disseminated intravascular coagulation.26Slofstra S.H. Groot A.P. Obdeijn M.H. Reitsma P.H. ten Cate H. Spek C.A. Gene expression profiling identifies C/EBPdelta as a candidate regulator of endotoxin-induced disseminated intravascular coagulation.Am J Respir Crit Care Med. 2007; 176: 602-609Crossref PubMed Scopus (14) Google Scholar However, the functions of C/EBPβ and C/EBPδ in acute lung inflammation remain poorly understood. C/EBPβ was recently shown to mediate inflammatory and innate immune responses in lung to cigarette smoke.27Didon L. Barton J.L. Roos A.B. Gaschler G.J. Bauer C.M. Berg T. Stampfli M.R. Nord M. Lung epithelial CCAAT/enhancer-binding protein-β is necessary for the integrity of inflammatory responses to cigarette smoke.Am J Respir Crit Care Med. 2011; 184: 233-242Crossref PubMed Scopus (26) Google Scholar C/EBPβ also reportedly played an essential role in bleomycin-induced pulmonary fibrosis.28Hu B. Ullenbruch M.R. Jin H. Gharaee-Kermani M. Phan S.H. An essential role for CCAAT/enhancer binding protein beta in bleomycin-induced pulmonary fibrosis.J Pathol. 2007; 211: 455-462Crossref PubMed Scopus (31) Google Scholar The role of C/EBPδ in lung inflammation and injury remains unknown. Herein, we identify C/EBPδ, but not C/EBPβ, as a critical regulator of inflammatory responses in lipopolysaccharide (LPS)–induced acute lung injury. Mouse alveolar macrophage–derived cell line, MH-S, was obtained from ATCC (Manassas, VA); cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.01 mol/L HEPES; and maintained in a humidified incubator at 37°C with 5% CO2. Enzyme-linked immunosorbent assay (ELISA) kits for mouse IL-6, tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-2, and keratinocyte cell–derived chemokine (KC) were obtained from R&D Systems (Minneapolis, MN). p38 Mitogen-activated protein kinase (MAPK) inhibitor VIII and p44/p42 inhibitor, U0126, were obtained from EMD Biosciences (Gibbstown, NJ). All procedures involving mice were approved by the Animal Care and Use Committee of Harvard Medical School (Boston, MA). Specific pathogen-free male C57BL/6 mice, aged 8 to 12 weeks, were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were anesthetized i.p. with 100 mg/kg ketamine HCl, followed by intratracheal instillation of 50 μL of LPS (1 mg/mL serotype 0111.B4; Sigma-Aldrich, St. Louis, MO) dissolved in PBS during inspiration. Negative control mice received 50 μL of PBS intratracheally. Unless otherwise indicated, 18 hours after LPS deposition, mice were exsanguinated and the pulmonary circulation was flushed with 1 mL of PBS via the pulmonary artery. The lungs were surgically dissected and immediately frozen in liquid nitrogen. The generation of C/EBPβ−/− and C/EBPδ−/− mice by homologous recombination was previously described.29Sterneck E. Tessarollo L. Johnson P.F. An essential role for C/EBPbeta in female reproduction.Genes Dev. 1997; 11: 2153-2162Crossref PubMed Scopus (338) Google Scholar, 30Sterneck E. Paylor R. Jackson-Lewis V. Libbey M. Przedborski S. Tessarollo L. Crawley J.N. Johnson P.F. Selectively enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/enhancer binding protein delta.Proc Natl Acad Sci U S A. 1998; 95: 10908-10913Crossref PubMed Scopus (127) Google Scholar Cebpb−/− mice and wild-type (WT) littermates were on a C57BL/6:Sv129 F1 hybrid background (to circumvent low mutant viability on each pure strain background), and Cebpd−/− animals and WT controls were on a C57Bl/6 background. Mice were sacrificed, and the lungs were perfused via the right ventricle with 3 mL of PBS. To measure myeloperoxidase (MPO) activity, whole lungs were homogenized in 50 mmol/L potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide and 5 mmol/L EDTA. The samples were sonicated for 1 minute and centrifuged at 9600 × g for 10 minutes. A total of 10 μL of the recovered supernatants was added to a 96-well plate, followed by the addition of 100 mmol/L potassium phosphate buffer containing 1.5 mol/L H2O2 and 167 μg/mL o-dianisidine dihydrochloride. The enzyme activity was determined by measuring the change in OD at 450 nm over 4.5 minutes using a 96-well plate reader. At 18 hours after LPS deposition, 1 mL of 10% buffered (pH 7.2) formalin was instilled into the lung via the trachea. The lungs were then surgically removed and further fixed in 10% buffered formalin solution for morphological assay by tissue sectioning and staining with H&E. At 18 hours after initiation of the acute lung injury, the thorax was opened and 0.8 mL of ice-cold, sterile PBS was instilled into the lung via a tracheal incision. The recovered bronchial alveolar lavage (BAL) fluid was centrifuged at 450 × g for 6 minutes, and the cell-free supernatants were stored at −20°C. Cell pellets were resuspended in 1 mL of HBSS containing 0.5% bovine serum albumin, and differential cell analyses were performed by Diff-Quik–stained cytospin preparations (Dade, Düdingen, Switzerland), counting 300 cells per slide in randomly selected high-powered fields (original magnification, ×1000). The supernatant was used for chemokine and cytokine measurements by sandwich ELISA. Mouse albumin levels in BAL fluid were measured using a mouse albumin ELISA kit obtained from Bethyl Laboratories (Montgomery, TX). Mice were anesthetized with 100 mg/kg i.p. ketamine HCl. A suspension of dichloromethylene diphosphonate (Cl2MDP) liposomes in PBS (10 μL of the liposome stock in a total volume of 50 μL) was administered intratracheally during inspiration. As a control, PBS liposomes were administered in a similar manner. All subsequent interventions were performed 24 hours after liposome instillation. Liposome-encapsulated clodronate was prepared as previously described.31Van Rooijen N. Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications.J Immunol Methods. 1994; 174: 83-93Crossref PubMed Scopus (1468) Google Scholar Mice receiving Cl2MDP liposomes showed >75% depletion of alveolar macrophages compared with mice receiving PBS liposomes. The administration of PBS liposomes did not reduce the number of alveolar macrophages. Nuclear extracts of whole lung tissues were prepared as previously described.21Browder W. Ha T. Chuanfu L. Kalbfleisch J.H. Ferguson Jr., D.A. Williams D.L. Early activation of pulmonary nuclear factor kappaB and nuclear factor interleukin-6 in polymicrobial sepsis.J Trauma. 1999; 46: 590-596Crossref PubMed Scopus (38) Google Scholar Briefly, frozen lungs were homogenized in 0.6% (v/v) Nonidet P-40, 150 mmol/L NaCl, 10 mmol/L HEPES (pH 7.9), 1 mmol/L EDTA, 0.5 mmol/L phenylmethylsulfonyl fluoride, 2.5 μg/mL leupeptin, 5 μg/mL antipain, and 5 μg/mL aprotinin. The homogenate was incubated on ice for 5 minutes and then centrifuged for 5 minutes at 5000 × g at 4°C. Proteins were extracted from the pelleted nuclei by incubation at 4°C with solution B [420 mmol/L NaCl, 20 mmol/L HEPES (pH 7.9), 1.2 mmol/L MgCl2, 0.2 mmol/L EDTA, 25% (v/v) glycerol, 0.5 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 2.5 μg/mL leupeptin, 5 μg/mL antipain, and 5 μg/mL aprotinin]. Nuclear debris was pelleted by centrifugation at 13,000 × g for 30 minutes at 4°C, and the supernatant extract was collected and stored at −80°C. Protein concentrations were determined by a Bio Rad protein assay using bovine serum albumin as a reference standard (Pierce Co, Rockford, IL). The electrophoretic mobility shift assay (EMSA) probes were double-stranded oligonucleotides containing a C/EBP consensus oligonucleotide (5′-TGCAGATTGCGCAATCTGCA-3′; Santa Cruz Biotechnology, Santa Cruz, CA). C/EBP probes were labeled with [32Gao H. Parkin S. Johnson P.F. Schwartz R.C. C/EBP gamma has a stimulatory role on the IL-6 and IL-8 promoters.J Biol Chem. 2002; 277: 38827-38837Crossref PubMed Scopus (40) Google ScholarP] ATP (3000 Ci/mmol at 10 mCi/mL) (Amersham Biosciences Corp, Sunnyvale, CA). DNA-binding reactions were performed at room temperature in a 25-μL reaction mixture containing 6 μL of nuclear extract (1 mg/mL in buffer C or solution B) and 5 μL of five times binding buffer [20% (w/v) Ficoll, 50 mmol/L HEPES (pH 7.9), 5 mmol/L EDTA, and 5 mmol/L dithiothreitol]. The remainder of the reaction mixture contained KCl at a final concentration of 50 mmol/L, Nonidet P-40 at a final concentration of 0.1%, 1 μg of poly(dI-dC), 200 pg of probe (unless otherwise noted), bromphenol blue at a final concentration of 0.06% (w/v), and water to a volume of 25 μL. Samples were electrophoresed through 5.5% polyacrylamide gels in one times TBE (90 mmol/L Tris base, 90 mmol/L boric acid, and 0.5 mmol/L EDTA) at 190 V for approximately 3.5 hours, dried under a vacuum, and exposed to X-ray film. For supershifts, nuclear extracts were pre-incubated with antibodies (1 to 2 μg) for 0.5 hours at 4°C before the binding reaction. The following antibodies were obtained from Santa Cruz Biotechnology, Inc.: C/EBPα, C/EBPβ, C/EBPδ, C/EBPε, C/EBPγ, and normal rabbit IgG. Transient small-interfering RNA (siRNA) transfections were performed by transfecting 1 × 106 to 2 × 106 MH-S cells with control siRNA or C/EBPδ siRNA (Santa Cruz Biotechnology) using Amaxa nucleofector kit V. After 12 hours, MH-S cells were treated with or without 100 ng/mL LPS (Sigma-Aldrich) for 6 hours. RNAs were harvested for RT-PCR to analyze down-regulation of C/EBPδ expression, or supernatants were collected for ELISAs. MH-S cells were transfected with the indicated reporter plasmids by using the Fugene6 Transfection Reagent (Roche, Indianapolis, IN). At 48 hours after transfection, cells were treated with or without 100 ng/mL LPS. After 4 hours, cells were lysed in Passive Lysis 5X Buffer (Promega, Madison, WI), and luciferase activity was measured. The mouse IL-6 promoter-reporter, the TNF-α promoter-reporter, and the C/EBPδ expression plasmid have been described in our previous publications.32Gao H. Parkin S. Johnson P.F. Schwartz R.C. C/EBP gamma has a stimulatory role on the IL-6 and IL-8 promoters.J Biol Chem. 2002; 277: 38827-38837Crossref PubMed Scopus (40) Google Scholar, 33Hu H.M. Baer M. Williams S.C. Johnson P.F. Schwartz R.C. Redundancy of C/EBP alpha, -beta, and -delta in supporting the lipopolysaccharide-induced transcription of IL-6 and monocyte chemoattractant protein-1.J Immunol. 1998; 160: 2334-2342PubMed Google Scholar Total RNAs were extracted from lungs with TRIzol (Invitrogen, Carlsbad, CA), according to the manufacturer's procedure. After isolation, total cellular RNA was incubated with RQ1 RNase-free DNase (Promega) to remove contaminating DNA. A total of 2 μg of total RNA was submitted to reverse transcription by using the Superscript II RNase H-Reverse Transcriptase (Invitrogen). PCR was performed with primers for the following: C/EBPä, 5′-CGCAGACAGTGGTGAGCTT-3′ (forward) and 5′-CTTCTGCTGCATCTCCTGGT-3′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GCCTCGTCTCATAGACAAGATG-3′ (forward) and 5′-CAGTAGACTCCACGACATAC-3′ (reverse). After a hot start for 5 minutes at 94°C, 28 to 33 cycles were used for amplification, with a melting temperature of 94°C, an annealing temperature of 60°C, and an extending temperature of 72°C, each for 1 minute, followed by a final extension at 72°C for 8 minutes. PCR was performed using different cycle numbers for all primers, to ensure that DNA was detected within the linear part of the amplifying curves for both primers. MH-S cells were lysed in radioimmune precipitation assay buffer. Samples containing 80 μg of protein were electrophoresed in a 12% polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane. Membranes were incubated with rabbit anti-C/EBPδ antibody (Santa Cruz Biotechnology), rabbit anti–phospho-p38 MAPK antibody (Cell Signaling, Danvers, MA), rabbit anti–phospho-p44/42 MAPK antibody (Cell Signaling), rabbit anti-p38 MAPK antibody (Cell Signaling), rabbit anti-p44/42 antibody (Cell Signaling), and rabbit anti–GAPDH antibody (Cell Signaling). After three washes in Tris-buffered saline with Tween, the membranes were incubated with a 1:5000 dilution of horseradish peroxidase–conjugated donkey anti-rabbit IgG (GE Healthcare, Woburn, MA). The membrane was developed by the enhanced chemiluminescence technique, according to the manufacturer's protocol (Thermo Fisher Scientific, Rockford, IL). All values were expressed as the mean ± SEM. Data sets were analyzed using the Student's t-test or a one-way analysis of variance, with individual group means being compared with the Student-Newman-Keuls multiple comparison test. The time course of C/EBP activation during LPS-induced lung injury was evaluated by EMSA, using nuclear extracts from whole lung obtained at various time points after the onset of lung injury. As shown in Figure 1A, at time 0, low levels of constitutive C/EBP DNA-binding species in whole lung nuclear extracts were observed. Increased C/EBP DNA binding was evident and strong by 3 to 24 hours. Antibody supershift assays were used to identify individual C/EBP family members present in the EMSA complexes. There are three DNA-binding species in the nuclear proteins of control-treated lungs (PBS): a low, but detectable, level of C/EBPα/β heterodimers, heterodimers between C/EBPβ liver-enriched activator protein and its short isoform, liver-enriched inhibitory protein, which is translated from an alternative start site in the same mRNA,34Descombes P. Schibler U. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein LIP, are translated from the same mRNA.Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (855) Google Scholar and liver-enriched inhibiting protein/liver-enriched inhibiting protein homodimers (Figure 1B). However, little C/EBPδ DNA binding was evident. In contrast, the binding activities of both C/EBPβ and C/EBPδ were significantly induced in LPS-injured lungs (Figure 1B). We next evaluated whether LPS induced expression of C/EBPβ and C/EBPδ at the protein level. Western blot analysis of lysates harvested over a time course after LPS deposition revealed increased abundance of both C/EBPβ and C/EBPδ during the 24-hour period (Figure 1C and data not shown). Thus, increased expression of the C/EBPβ and C/EBPδ proteins coincided with their enhanced DNA-binding activity in the lung. To localize the expression of C/EBPδ protein, we performed immunohistochemical staining in lung sections after LPS administration. We found strong staining for C/EBPδ protein in LPS-injured lung, especially in bronchiolar and alveolar epithelium (data not shown). We further performed immunocytostaining experiments using cells in BAL fluids harvested from lungs after LPS administration. Strong C/EBPδ expression was detectable in alveolar macrophages (data not shown). A previous study demonstrated that alveolar macrophage was a critical component for initiation of the LPS-induced innate immune response in the lung.35Koay M.A. Gao X. Washington M.K. Parman K.S. Sadikot R.T. Blackwell T.S. Christman J.W. Macrophages are necessary for maximal nuclear factor-kappa B activation in response to endotoxin.Am J Respir Cell Mol Biol. 2002; 26: 572-578Crossref PubMed Scopus (114) Google Scholar We sought to determine the role of macrophages in regulating C/EBP activation in whole lung tissues in response to LPS. Nuclear extracts from whole lungs harvested 3 hours after the onset of injury were analyzed by EMSA. As shown in Figure 1D, strong LPS-induced C/EBP activation in the lung occurred in mice pretreated with PBS liposomes. In contrast, mice pretreated with Cl2MDP liposomes showed significantly reduced lung C/EBP activation after LPS instillation (Figure 1D), suggesting that alveolar macrophages played a critical role in lung C/EBP activation induced by LPS. By using systems biology approaches, Litvak et al36Litvak V. Ramsey S.A. Rust A.G. Zak D.E. Kennedy K.A. Lampano A.E. Nykter M. Shmulevich I. Aderem A. Function of C/EBPdelta in a regulatory circuit that discriminates between transient and persistent TLR4-induced signals.Nat Immunol. 2009; 10: 437-443Crossref PubMed Scopus (227) Google Scholar recently reported that C/EBPδ played a critical role in a regulatory circuit that discriminated between transient and persistent Toll-like receptor (TLR) 4–induced signals. Nevertheless, the role of C/EBPδ in acute lung injury remained largely unknown. To examine whether C/EBPδ was involved in lung injury after LPS deposition, we first measured leakage of mouse albumin into the lungs of wild-type and C/EBPδ-deficient mice. Endogenous mouse albumin levels in BAL fluids were determined by ELISA.37Sun L. Guo R.F. Gao H. Sarma J.V. Zetoune F.S. Ward P.A. Attenuation of IgG immune complex-induced acute lung injury by silencing C5aR in lung epithelial cells.FASEB J. 2009; 23: 3808-3818Crossref PubMed Scopus (38) Google Scholar As shown in Figure 2A, albumin levels in BAL fluids were significantly increased in WT mice that received intratracheal administration of LPS. However, the BAL fluid albumin levels in C/EBPδ-deficient mice remained at nearly background levels (Figure 2A). We also examined MPO content to evaluate neutrophil accumulation in the lung. As shown in Figure 2B, C/EBPδ deficiency alone did not cause a change in MPO activity in the lung. LPS administration led to increased MPO activity in WT mice, whereas MPO content in LPS-treated C/EBPδ-deficient mice was modestly, but significantly (P < 0.05), reduced relative to WT mice (Figure 2B). We next evaluated the effects of C/EBPδ deficiency on leukocyte content in BAL fluids from LPS-injured lung. As shown in Figure 2, C and D, injured lungs from WT and C/EBPδ-deficient mice contained mostl
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