Title: TLR4 Activity Is Required in the Resolution of Pulmonary Inflammation and Fibrosis after Acute and Chronic Lung Injury
Abstract: Pulmonary fibrosis is an inflammation-driven lung disease with a poor prognosis and no cure. Here we report that basal toll-like receptor 4 (TLR4) activity is critical for the resolution of acute and chronic inflammation and pulmonary fibrosis in mouse models of lung injury. We found that genetic or pharmacologic inhibition of TLR4 exacerbates bleomycin-induced pulmonary inflammation, fibrosis, dysfunction, and animal death through promoting formation of an immunosuppressive tissue microenvironment and attenuating autophagy-associated degradation of collagen and cell death in the fibrotic lung tissues. In contrast, pharmacologic activation of TLR4 resulted in a quick resolution of acute inflammation, reversed the established pulmonary fibrosis, improved lung function, and rescued mice from death. Similarly, blocking TLR4 impaired the resolution of silica-induced chronic inflammation and fibrosis. Importantly, altering autophagic activity could reverse the TLR4-regulated lung inflammation, fibrosis, dysfunction, and animal death. Rapamycin, an autophagy activator, reversed the effects of TLR4 antagonism. In contrast, inhibition of autophagy by 3-methyladenine reversed the proresolving and antifibrotic roles of TLR4 agonists and increased animal death. These results not only highlight a pivotal role for TLR4-mediated basal immunity, particularly autophagic activity, in the proresolution of inflammation and fibrosis after chemical-induced lung injury but also provide proof for the concept for activating TLR4 signaling, particularly TLR4-mediated autophagy, as a novel therapeutic strategy against chronic fibroproliferative diseases that are unresponsive to current therapy. Pulmonary fibrosis is an inflammation-driven lung disease with a poor prognosis and no cure. Here we report that basal toll-like receptor 4 (TLR4) activity is critical for the resolution of acute and chronic inflammation and pulmonary fibrosis in mouse models of lung injury. We found that genetic or pharmacologic inhibition of TLR4 exacerbates bleomycin-induced pulmonary inflammation, fibrosis, dysfunction, and animal death through promoting formation of an immunosuppressive tissue microenvironment and attenuating autophagy-associated degradation of collagen and cell death in the fibrotic lung tissues. In contrast, pharmacologic activation of TLR4 resulted in a quick resolution of acute inflammation, reversed the established pulmonary fibrosis, improved lung function, and rescued mice from death. Similarly, blocking TLR4 impaired the resolution of silica-induced chronic inflammation and fibrosis. Importantly, altering autophagic activity could reverse the TLR4-regulated lung inflammation, fibrosis, dysfunction, and animal death. Rapamycin, an autophagy activator, reversed the effects of TLR4 antagonism. In contrast, inhibition of autophagy by 3-methyladenine reversed the proresolving and antifibrotic roles of TLR4 agonists and increased animal death. These results not only highlight a pivotal role for TLR4-mediated basal immunity, particularly autophagic activity, in the proresolution of inflammation and fibrosis after chemical-induced lung injury but also provide proof for the concept for activating TLR4 signaling, particularly TLR4-mediated autophagy, as a novel therapeutic strategy against chronic fibroproliferative diseases that are unresponsive to current therapy. Fibroproliferative diseases are characterized by chronic inflammatory response and tissue fibrosis and often result in severe disruption of organ structure and function. A fatal outcome is possible after tissue injury caused by microbial infection, chemical agents, stress, and drugs.1Rosenbloom J. Castro S.V. Jimenez S.A. Narrative review: fibrotic diseases: cellular and molecular mechanisms and novel therapies.Ann Intern Med. 2010; 152: 159-166Crossref PubMed Google Scholar, 2du Bois R.M. Strategies for treating idiopathic pulmonary fibrosis.Nat Rev Drug Discov. 2010; 9: 129-140Crossref PubMed Scopus (209) Google Scholar Despite recent improved insights into disease pathogenesis and identification of new molecular targets, the precise pathologic mechanisms and, particularly, an effective drug therapy for fibroproliferative diseases has yet to be identified.1Rosenbloom J. Castro S.V. Jimenez S.A. Narrative review: fibrotic diseases: cellular and molecular mechanisms and novel therapies.Ann Intern Med. 2010; 152: 159-166Crossref PubMed Google Scholar, 3de Andrade J.A. Thannickal V.J. Innovative approaches to the therapy of fibrosis.Curr Opin Rheumatol. 2009; 21: 649-655Crossref PubMed Scopus (22) Google Scholar, 4Boucher R.C. Idiopathic pulmonary fibrosis–a sticky business.N Engl J Med. 2011; 364: 1560-1561Crossref PubMed Scopus (35) Google Scholar Indeed, based on observations from clinical trials and animal studies, especially the fact that immunosuppressive and anti-inflammatory agents are relatively ineffective against tissue fibrosis, it is increasingly questioned whether inflammation is a major component of established fibrotic pathologic conditions.2du Bois R.M. Strategies for treating idiopathic pulmonary fibrosis.Nat Rev Drug Discov. 2010; 9: 129-140Crossref PubMed Scopus (209) Google Scholar, 5Datta A. Scotton C.J. Chambers R.C. Novel therapeutic approaches for pulmonary fibrosis.Br J Pharmacol. 2011; 163: 141-172Crossref PubMed Scopus (167) Google Scholar Moreover, recent clinical trials for idiopathic pulmonary fibrosis (IPF) failed to identify unequivocally a survival benefit after testing a number of agents with various mechanisms of action, including pirfenidone, bosentan, etanercept, interferons, N-acetylcysteine, warfarin, the protein kinase inhibitor imatinib, and the phosphodiesterase-5 inhibitor sildenafil.6Perazella M.A. Markowitz G.S. Drug-induced acute interstitial nephritis.Nat Rev Nephrol. 2010; 6: 461-470Crossref PubMed Scopus (224) Google Scholar, 7Zisman D.A. Schwarz M. Anstrom K.J. Collard H.R. Flaherty K.R. Hunninghake G.W. A controlled trial of sildenafil in advanced idiopathic pulmonary fibrosis.N Engl J Med. 2010; 363: 620-628Crossref PubMed Scopus (470) Google Scholar As a result, there is an urgent need to develop safe and effective therapeutic agents for fibroproliferative diseases. Despite controversy, we believe that acute and chronic inflammation plays a critical role in the initiation and progression of tissue fibrosis after acute tissue injury caused by a variety of insults.2du Bois R.M. Strategies for treating idiopathic pulmonary fibrosis.Nat Rev Drug Discov. 2010; 9: 129-140Crossref PubMed Scopus (209) Google Scholar, 8Strieter R.M. Mehrad B. New mechanisms of pulmonary fibrosis.Chest. 2009; 136: 1364-1370Crossref PubMed Scopus (224) Google Scholar The inflammation is elicited through diverse molecules with pathogen- or damage-associated molecular patterns interacting with pattern recognition receptors, which include toll-like receptors (TLRs), RIG-1–like receptors, NOD-like receptors, and C-type lectin receptors.9Delgado M. Singh S. De Haro S. Master S. Ponpuak M. Dinkins C. Ornatowski W. Vergne I. Deretic V. Autophagy and pattern recognition receptors in innate immunity.Immunol Rev. 2009; 227: 189-202Crossref PubMed Scopus (145) Google Scholar The properties, duration, and intensity of the inflammatory response, rather than inflammation per se, direct the development and progression of tissue fibrosis.10Wynn T.A. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases.J Clin Invest. 2007; 117: 524-529Crossref PubMed Scopus (1107) Google Scholar Our previous studies have indicated that TLR2 mediates bleomycin (BLM)-induced acute lung injury, pulmonary inflammation, and fibrosis and that blocking TLR2 activity promotes the resolution of pulmonary inflammation and fibrosis.11Yang H.Z. Cui B. Liu H.Z. Chen Z.R. Yan H.M. Hua F. Hu Z.W. Targeting TLR2 attenuates pulmonary inflammation and fibrosis by reversion of suppressive immune microenvironment.J Immunol. 2009; 182: 692-702Crossref PubMed Scopus (82) Google Scholar In most situations, TLR4 and TLR2 play similar roles in the mediation of inflammatory response because they share many common ligands and a common inflammatory signaling pathway.12Williams A.S. Leung S.Y. Nath P. Khorasani N.M. Bhavsar P. Issa R. Mitchell J.A. Adcock I.M. Chung K.F. Role of TLR2, TLR4, and MyD88 in murine ozone-induced airway hyperresponsiveness and neutrophilia.J Appl Physiol. 2007; 103: 1189-1195Crossref PubMed Scopus (99) Google Scholar Moreover, Imai et al13Imai Y. Kuba K. Neely G.G. Yaghubian-Malhami R. Perkmann T. van Loo G. Ermolaeva M. Veldhuizen R. Leung Y.H. Wang H. Liu H. Sun Y. Pasparakis M. Kopf M. Mech C. Bavari S. Peiris J.S. Slutsky A.S. Akira S. Hultqvist M. Holmdahl R. Nicholls J. Jiang C. Binder C.J. Penninger J.M. Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury.Cell. 2008; 133: 235-249Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar recently reported that acute lung injury caused by multiple lung pathogens is triggered by the signaling of oxidized phospholipids through activation of TLR4. Seki et al14Seki E. De Minicis S. Osterreicher C.H. Kluwe J. Osawa Y. Brenner D.A. Schwabe R.F. TLR4 enhances TGF-β signaling and hepatic fibrosis.Nat Med. 2007; 13: 1324-1332Crossref PubMed Scopus (1484) Google Scholar found that activation of TLR4 enhances transforming growth factor (TGF)-β signaling in the development of hepatic fibrosis. We and others observed that TLR4 mediates pressure overload–induced cardiovascular fibrosis and remodeling through activation of a chronic inflammatory response.15Timmers L. Sluijter J.P. van Keulen J.K. Hoefer I.E. Nederhoff M.G. Goumans M.J. Doevendans P.A. van Echteld C.J. Joles J.A. Quax P.H. Piek J.J. Pasterkamp G. de Kleijn D.P. Toll-like receptor 4 mediates maladaptive left ventricular remodeling and impairs cardiac function after myocardial infarction.Circ Res. 2008; 102: 257-264Crossref PubMed Scopus (281) Google Scholar, 16Liu Y.Y. Cai W.F. Yang H.Z. Cui B. Chen Z.R. Liu H.Z. Yan J. Jin W. Yan H.M. Xin B.M. Yuan B. Hua F. Hu Z.W. Bacillus Calmette-Guerin and TLR4 agonist prevent cardiovascular hypertrophy and fibrosis by regulating immune microenvironment.J Immunol. 2008; 180: 7349-7357PubMed Google Scholar We therefore hypothesized that inhibition of TLR4 signaling was able to protect mice from lung injury, acute and chronic inflammation, lung dysfunction, and pulmonary fibrosis after acute or chronic lung injury caused by chemical agents. Unexpectedly, our studies demonstrated an opposite effect of TLR4 antagonism on the BLM- or silica-induced mouse models of acute or chronic lung injury. We found that the basal TLR4 activity, particularly the TLR4-mediated activation of autophagy, is crucial to maintain an optimal tissue environment and to promote the resolution of acute or chronic inflammation and fibrogenesis after acute or chronic lung injury. Our results suggest that the immunostimulants eliciting autophagic activity, such as TLR4 agonists, instead of immunosuppressive agents, may be a promising therapeutic strategy for the treatment of devastating fibroproliferative diseases such as IPF. Ec-LPS (purified from Escherichia coli 0111: B4 strain, TLR4 agonist) [Lipopolysaccaride (LPS)-EB Ultrapure] was obtained from InvivoGen (San Diego, CA). Alexa Fluor 488-, Alexa Fluor 555-, or Alexa Fluor 647-conjugated anti-mouse CD4, CD25, FoxP3, PDCA-1, F4/80, CD206, CD11b, and TLR4 (clone UT41) antibodies were purchased from eBioscience (San Diego, CA). An annexin V/propidium iodide (PI) apoptosis assay kit was obtained from SBA Sciences (Turku, Finland). TLR4-neutralizing monoclonal antibody (mAb) (clone MTS510, #117608) was obtained from BioLegend (San Diego, CA). The blocking effects of the TLR4-neutralizing mAb were verified as described previously.17Yang H.Z. Cui B. Liu H.Z. Mi S. Yan J. Yan H.M. Hua F. Lin H. Cai W.F. Xie W.J. Lv X.X. Wang X.X. Xin B.M. Zhan Q.M. Hu Z.W. Blocking TLR2 activity attenuates pulmonary metastases of tumor.PLoS ONE. 2009; 4: e6520Crossref PubMed Scopus (84) Google Scholar The anti–α-smooth muscle actin (SMA), β-actin, glyceraldehyde-3-phosphate dehydrogenase, apoptosis signal-regulating kinase 1 (ASK1), phospho-ASK1, active (cleaved) caspase 3, p38 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK, JNK, phospho-JNK, ERK, phospho-ERK, β-tubulin, NF-κB, phospho–NF-κB, mammalian target of rapamycin (mTOR), phospho-mTOR, Akt, and phospho-Akt antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA). The anti-LC3B, Beclin1, PI3K3C, and LAMP1 antibodies were from Abcam (Cambridge, UK). ECL Plus Western blotting detection reagents were from Amersham Biosciences (Piscataway, NJ). Bleomycin (BLM, 1 U/mg) was from Nippon Kayaku (Tokyo, Japan). Rapamycin and 3-methyladenine (3-MA) were from Sigma-Aldrich (Shanghai, China). Pulmonary hydroxyproline levels were evaluated with a commercially available kit (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China). The endotoxin level in the solutions of BLM and neutralizing Abs was <0.01 ng/mL, as tested by the Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). The reactive oxygen species (ROS) testing kit was purchased from Genmed Scientifics Inc. (Shanghai, China). All other materials were purchased from standard commercial sources. TLR4-deficient (TLR4−/−) and corresponding wild-type (WT) mice (C3H) were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). C57BL/6J mice (18 g ± 1 g, 6 to 8 weeks old) were obtained from the Vital River Lab Animal Technology, Co. Ltd. (Beijing, China). The animals were housed in a facility with a 12-hour light/12-hour dark cycle and were given free access to water and standard rodent chow. The room was kept free of specific pathogens. The care and treatment of experimental animals was in accordance with institutional guidelines at the Experimental Animal Center of the Chinese Academy of Medical Sciences. To generate the animal model of acute inflammation and pulmonary fibrosis, mice were anesthetized with i.p. pentobarbital (50 mg/kg) (Merck, Woodbridge, NJ) and injected intratracheally with BLM (3.0 mg/kg) in 50 μL of LPS-free saline using an insulin syringe as previously described.11Yang H.Z. Cui B. Liu H.Z. Chen Z.R. Yan H.M. Hua F. Hu Z.W. Targeting TLR2 attenuates pulmonary inflammation and fibrosis by reversion of suppressive immune microenvironment.J Immunol. 2009; 182: 692-702Crossref PubMed Scopus (82) Google Scholar To determine the effects of targeting TLR4 on established pulmonary fibrosis,18Moeller A. Ask K. Warburton D. Gauldie J. Kolb M. The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis.Int J Biochem Cell Biol. 2008; 40: 362-382Crossref PubMed Scopus (677) Google Scholari.v. TLR4-neutralizing or isotype-matched antibody (200 μg/kg in 200 μL saline) was injected on the tenth and 17th days after BLM instillation; i.p. TLR4 agonist Ec-LPS (10 mg/kg/day) was injected once daily from day 10 to day 17 after BLM instillation. To determine the roles of autophagy in TLR4-regulated pulmonary fibrosis, i.p anti-TLR4 antibody was given with or without rapamycin (10 mg/kg/day from day 9 to day 17 after BLM instillation), whereas i.p. Ec-LPS was given with or without 3-MA (30 mg/kg/day from day 9 to day 17 after BLM instillation). Four weeks after BLM administration, the mice were sacrificed by excessive anesthesia for the collection of single-cell suspensions, bronchoalveolar lavage fluid (BALF), and lungs at the indicated time points. The lungs were excised and fixed or frozen for morphologic evaluation, analysis of cytokine and chemokine expression, or the measurement of hydroxyproline content. To generate a mouse model of chronic inflammation and fibrosis, C57BL/6J mice were administered SiO2 (2.5 mg/mouse) intratracheally as described previously,11Yang H.Z. Cui B. Liu H.Z. Chen Z.R. Yan H.M. Hua F. Hu Z.W. Targeting TLR2 attenuates pulmonary inflammation and fibrosis by reversion of suppressive immune microenvironment.J Immunol. 2009; 182: 692-702Crossref PubMed Scopus (82) Google Scholar and a TLR4-neutralizing or isotype-matched antibody (200 μg/kg) was administered intravenously once a week from day 14 after SiO2 treatment. Two months after SiO2 administration, animals were used for measuring lung function or sacrificed by excessive anesthesia for the collection of lungs and BALF for the indicated analysis. At the end of the experiment, the mice were anesthetized, underwent tracheostomy, and were placed in a forced pulmonary maneuver system (Buxco Research Systems, Wilmington, NC) as described previously.19Vanoirbeek J.A. Rinaldi M. De Vooght V. Haenen S. Bobic S. Gayan-Ramirez G. Hoet P.H. Verbeken E. Decramer M. Nemery B. Janssens W. Noninvasive and invasive pulmonary function in mouse models of obstructive and restrictive respiratory diseases.Am J Respir Cell Mol Biol. 2009; 42: 96-104Crossref PubMed Scopus (237) Google Scholar Lung compliance (Cchord) and functional residual capacity (FRC) were measured. All data reflect measurements from at least 5 mice per group. Microcomputed tomography (micro-CT) images were generated as described previously.20Shofer S. Badea C. Qi Y. Potts E. Foster W.M. Johnson G.A. A micro-CT analysis of murine lung recruitment in bleomycin-induced lung injury.J Appl Physiol. 2008; 105: 669-677Crossref PubMed Scopus (18) Google Scholar Animals were anesthetized with i.p. chloral hydrate (400 mg/kg) to achieve prolonged sedation. The animals were intubated perorally, placed on an acrylic cradle, and suspended vertically for imaging. X-ray parameters were as follows: 80 kilovolts (peak) [kV(p)], 220 mA, and 12-msec exposure per projection. Five hundred ten projections were acquired at a 0.725-degree increment between projections for a total rotation angle of 370 degrees. Data were reconstructed with weighting according to Parker as isotropic 512 × 512 × 512 arrays with effective digital sampling of 100 μm along all three axes. Reconstructed images were converted to Hounsfield units (HU) by scaling air selected from a region outside the animal to −1000 HU and water to 0 HU using a water-filled phantom secured to the abdomen of each animal via a conversion file written for MATLAB (The MathWorks, Natick, MA). BALF was collected and processed as previously described.11Yang H.Z. Cui B. Liu H.Z. Chen Z.R. Yan H.M. Hua F. Hu Z.W. Targeting TLR2 attenuates pulmonary inflammation and fibrosis by reversion of suppressive immune microenvironment.J Immunol. 2009; 182: 692-702Crossref PubMed Scopus (82) Google Scholar In brief, the trachea was exposed through a midline incision and cannulated with a sterile 22-gauge Abbocath-T catheter. Bilateral BAL was performed by instilling two 0.5-mL aliquots of sterile saline. Approximately 0.9 to 1.0 mL of BALF was retrieved per mouse. The cellular viabilities assessed by 0.4% trypan blue exclusion were greater than 99%. The total cell count of the BALF was assessed with a hemocytometer (Neubauer Zählkammer, Gehrden, Germany). The cells were then washed, resuspended, and analyzed by a hematology analyzer or stained with PE-Cy5–, APC- or fluorescein isothiocyanate (FITC)-conjugated antibodies for flow cytometry. The concentrations of interferon (IFN)-γ, TGF-β1, interleukin (IL)-4, IL-5, IL-10, and IL-13 in BALF were measured with enzyme-linked immunoassay kits following the manufacturer's instructions. The immune cells in BALF were analyzed using flow cytometry as described previously.21Traidl-Hoffmann C. Mariani V. Hochrein H. Karg K. Wagner H. Ring J. Mueller M.J. Jakob T. Behrendt H. Pollen-associated phytoprostanes inhibit dendritic cell interleukin-12 production and augment T helper type 2 cell polarization.J Exp Med. 2005; 201: 627-636Crossref PubMed Scopus (245) Google Scholar In brief, BALF was harvested, washed, and suspended in cold phosphate-buffered saline containing 3% fetal bovine serum, and 0.02% NaN3. The cells were then incubated with a mixture of rat and mouse IgG (1:1) to block nonspecific binding, followed by serial incubations with Alexa Fluor 488-, Alexa Fluor 568-, and/or Alexa Fluor 647-conjugated mAb for 1 hour at 4°C. Isotype-matched mAbs were used in control samples. After incubation, Foxp3+ CD4+ CD25+ regulatory T cells (Foxp3+ Tregs), PDCA+ plasmacytoid dendritic cells (pDCs), CD11b+ F4/80+ CD206− M1 macrophages, and CD11b+ F4/80+ CD206+ M2 macrophages were analyzed using CellQuest software (Becton Dickinson, CA). At the end of the experiment, the lungs were rapidly excised, fixed with 4% paraformaldehyde, and embedded in paraffin for histopathologic examination. Tissue sections (5-μm thick) were prepared and stained with H&E or Masson's trichrome. The grades of pulmonary inflammation and fibrosis were analyzed by a professional pathologist, who was blinded for groups. The average integrated optical density of the collagen deposition was determined by Image-Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD) in 10 randomly chosen regions per tissue sample at a magnification of ×200. Collagen deposition was also determined by measuring the total hydroxyproline content of the lungs according to Reddy's method, with modifications.11Yang H.Z. Cui B. Liu H.Z. Chen Z.R. Yan H.M. Hua F. Hu Z.W. Targeting TLR2 attenuates pulmonary inflammation and fibrosis by reversion of suppressive immune microenvironment.J Immunol. 2009; 182: 692-702Crossref PubMed Scopus (82) Google Scholar In brief, the lungs were hydrolyzed with 2.5 N NaOH at 120°C 0.1 kPa for 40 minutes. After neutralization with hydrochloric acid, the hydrolysates were diluted with distilled water. Hydroxyproline in the hydrolysates was assessed calorimetrically at 550 nm with p-dimethylaminobenzaldehyde. Results are presented as micrograms per milligrams of wet lung. Lung sections (5-μm thick) were prepared as described earlier, and apoptosis was detected by TUNEL using the ApopTag Peroxidase in Situ Apoptosis Detection Kit (Millipore, Billerica, MA). The lung sections were stained and TUNEL-positive cells were counted according to the manufacturer's protocol.22Hamacher-Brady A. Brady N.R. Logue S.E. Sayen M.R. Jinno M. Kirshenbaum L.A. Gottlieb R.A. Gustafsson A.B. Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy.Cell Death Differ. 2007; 14: 146-157Crossref PubMed Scopus (512) Google Scholar JHU-1 cells were obtained from ATCC (CRL-2785; Manassas, VA) and cultured in medium without serum and treated with FITC-labeled collagen for 30 minutes as previously described.23Atabai K. Jame S. Azhar N. Kuo A. Lam M. McKleroy W. Dehart G. Rahman S. Xia D.D. Melton A.C. Wolters P. Emson C.L. Turner S.M. Werb Z. Sheppard D. Mfge8 diminishes the severity of tissue fibrosis in mice by binding and targeting collagen for uptake by macrophages.J Clin Invest. 2009; 119: 3713-3722Crossref PubMed Scopus (168) Google Scholar The cells were then rinsed with phosphate-buffered saline three times. The cells were then cultured in microplate Alomar blue assay medium and treated with the indicated agents for 3 hours. Cells were washed, collected, and suspended in cold phosphate-buffered saline containing 3% fetal bovine serum and 0.02% NaN3. Fifty thousands cells were analyzed using CellQuest software (BD Biosciences). Total ROS production in the lungs was measured using 2′,7′-dichlorofluorescein diacetate according to the manufacturer's protocol.24Chen J.X. Stinnett A. Critical role of the NADPH oxidase subunit p47phox on vascular TLR expression and neointimal lesion formation in high-fat diet-induced obesity.Lab Invest. 2008; 88: 1316-1328Crossref PubMed Scopus (45) Google Scholar Data were analyzed with the LSM510 software (Carl Ziess, Göttingen Germany) and presented as the fluorescence intensity of ROS (arbitrary units). Autophagy was evaluated by immunofluorescence microscopy, electron microscopy, or immunoblot as described previously.25Xu Y. Jagannath C. Liu X.D. Sharafkhaneh A. Kolodziejska K.E. Eissa N.T. Toll-like receptor 4 is a sensor for autophagy associated with innate immunity.Immunity. 2007; 27: 135-144Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar In the immunofluorescence microscopy experiments, autophagy was evaluated by examining the punctate forms of the autophagy marker LC3 and the co-localization of LC3 and the lysosome marker LAMP1.26Korolchuk V.I. Saiki S. Lichtenberg M. Siddiqi F.H. Roberts E.A. Imarisio S. Jahreiss L. Sarkar S. Futter M. Menzies F.M. O'Kane C.J. Deretic V. Rubinsztein D.C. Lysosomal positioning coordinates cellular nutrient responses.Nat Cell Biol. 2011; 13: 453-460Crossref PubMed Scopus (584) Google Scholar Quantification of autophagy was performed based on the percentage of cells with LC3-containing punctate dots. In electron microscopy experiments, autophagy was evaluated by observing the typical double-membrane vesicles and quantified by the number of autophagic vacuoles per cross-sectioned cell. In all experiments, six samples were counted per experimental condition.11Yang H.Z. Cui B. Liu H.Z. Chen Z.R. Yan H.M. Hua F. Hu Z.W. Targeting TLR2 attenuates pulmonary inflammation and fibrosis by reversion of suppressive immune microenvironment.J Immunol. 2009; 182: 692-702Crossref PubMed Scopus (82) Google Scholar Proteins were extracted from lung tissues using a Qproteome Plasma Nuclei Protein Kit (Qiagen Inc., Valencia, CA). Protein concentrations were determined with Coomassie Plus reagent. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot were conducted as described previously.17Yang H.Z. Cui B. Liu H.Z. Mi S. Yan J. Yan H.M. Hua F. Lin H. Cai W.F. Xie W.J. Lv X.X. Wang X.X. Xin B.M. Zhan Q.M. Hu Z.W. Blocking TLR2 activity attenuates pulmonary metastases of tumor.PLoS ONE. 2009; 4: e6520Crossref PubMed Scopus (84) Google Scholar Protocols for immunofluorescence microscopy were described previously.27Salazar M. Carracedo A. Salanueva I.J. Hernandez-Tiedra S. Lorente M. Egia A. Vazquez P. Blazquez C. Torres S. Garcia S. Nowak J. Fimia G.M. Piacentini M. Cecconi F. Pandolfi P.P. Gonzalez-Feria L. Iovanna J.L. Guzman M. Boya P. Velasco G. Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells.J Clin Invest. 2009; 119: 1359-1372Crossref PubMed Scopus (552) Google Scholar Lung sections (5-μm thick) were prepared and stained with corresponding primary antibodies overnight at 4°C. The sections were then washed twice and incubated with fluorochrome-labeled secondary antibodies (1:200) for 30 minutes followed by three washes. Images were captured with a Leica SP2 confocal microscope (Leica Microsystems Inc., Exton, PA) and analyzed with Leica confocal software. The autophagosomes were evaluated by the co-expression of LC3 and LAMP1. Autophagic cell death was determined by a combination of LC3 and TUNEL, and autophagic cell apoptosis was determined by the combination of LC3 and active caspase 3. Data are presented as mean ± SD. Statistical analysis was performed with one-way analysis of variance, in which a threshold P value was set at 0.05, followed by Tukey-Kramer's or Dunnett's post hoc multiple comparison tests. Survival rates were analyzed by the Kaplan-Meier method. We have found previously that targeting TLR2 markedly attenuates the BLM-induced lung injury, inflammation, and fibrosis via reversal of the BLM-induced immunosuppressive tissue environment.11Yang H.Z. Cui B. Liu H.Z. Chen Z.R. Yan H.M. Hua F. Hu Z.W. Targeting TLR2 attenuates pulmonary inflammation and fibrosis by reversion of suppressive immune microenvironment.J Immunol. 2009; 182: 692-702Crossref PubMed Scopus (82) Google Scholar Because TLR4 plays a pivotal role in the initiation and orchestration of the inflammatory response to a variety of lung pathogens, we wondered if blocking TLR4 also attenuates BLM-induced pulmonary inflammation and fibrosis. Unexpectedly, we found that TLR4 deficiency notably increased the levels of lung injury, pulmonary inflammation, and fibrosis after BLM treatment. TLR4-deficient (TLR4−/−) mice exhibited markedly enhanced pulmonary fibrosis, as indicated by increased collagen deposition, α-SMA expression, and hydroxyproline content in the lungs (Figure 1A–E). Also, TLR4 deficiency significantly increased pulmonary inflammation (Figure 1, A and B) and infiltration of inflammatory cells, including total white blood cells, lymphocytes, neutrophils, basophils, eosinophils, and macrophages in BALF (see Supplemental Table S1 at http://ajp.amjpathol.org). Consistent with these pulmonary structural changes and inflammatory status, BLM-treated WT animals presented a significant reduction in lung function. TLR4 deficiency caused a further decline in lung function, as indicated by decreases in Cchord and FRC (Figure 1F), as well as other parameters of lung function (see Supplemental Figure S1 at http://ajp.amjpathol.org). Additionally, micro-CT was used to obtain three-dimensional information about lung structure. The BLM-treated TLR4−/− mice exhibited a significant reduction in lung density, as represented by a reduction in HU values, compared with BLM-treated WT mice (Figure 1, G and H). As a result, TLR4 deficiency reduced the survival rate of BLM-treated mice (Figure 1I). After acute lung injury, a Th1-predominant immune response is necessary for the resolution of inflammation and fibrosis, whereas a Th2-predominant immune response contributes to the development of chronic inflammation and fibrosis.11Yang H.Z. Cui B. Liu H.Z. Chen Z.R. Yan H.M. Hua F. Hu Z.W. Targeting TLR2 attenuates pulmonary inflammation and fibrosis by reversion of suppressive immune microenvironment.J Immunol. 2009; 182: 692-702Crossref PubMed Scopus (8