Toll like receptor 2 mediates bleomycin-induced acute lung injury ...

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药学学报 Acta Pharmaceutica Sinica 2010, 45 (8): 976−986

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·研究论文·

Toll like receptor 2 mediates bleomycin-induced acute lung injury, inflammation and fibrosis in mice LIU Han-zhi, YANG Hong-zhen, MI Su, CUI Bing, HUA Fang, HU Zhuo-wei* (Molecular Immunology and Pharmacology Laboratory, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China)

Abstract: Anti-cancer drug bleomycin (BLM) can cause acute lung injury (ALI) which often results in pulmonary fibrosis due to a failure of resolving acute inflammatory response. The aim of this study is to investigate whether toll-like receptor (TLR) 2 mediates BLM-induced ALI, inflammation and fibrosis. BLM-induced dendritic cells (DCs) maturation was analyzed by flow cytometry and cytokine secretion was detected by the ELISA method. The expression and activity of p38 and ERK MAPK were determined with Western blotting. The roles of TLR2 in ALI, inflammation and fibrosis were investigated in C57BL/6 mice administered intratracheally with BLM. The results demonstrated that BLM-administered mice had higher expression of TLR2 (P < 0.001) and its signaling molecules. Blocking TLR2 significantly inhibited the maturation of DCs and reversed BLM-stimulated secretion of cytokines in DCs, such as IL-6 (P < 0.001), IL-17 (P < 0.05) and IL-23 (P < 0.05). TLR2 inhibition attenuated BLM-induced increase of inflammatory cells in bronchoalveolar lavage fluid (BALF), and reversed the immunosuppressive microenvironment by enhancing TH1 response (P < 0.05) and inhibiting TH2 (P < 0.001), Treg (P < 0.01) and TH17 (P < 0.01) responses. Importantly, blocking TLR2 in vivo significantly protected BLM-administered mice from pulmonary injury, inflammation and fibrosis and subsequently increased BLM-induced animal survival (from 50% to 92%). Therefore, TLR2 is a novel potential target for ALI and pulmonary fibrosis. Key words: TLR2; bleomycin; dendritic cell; pulmonary inflammation; acute lung injury; fibrosis CLC number: R963 Document code: A Article ID: 0513-4870 (2010) 08-0976-11

Toll 样受体 2 介导博莱霉素诱导的急性肺损伤、炎症与纤维化刘含智, 杨红振, 米

粟, 崔

冰, 花

芳, 胡卓伟*

(中国医学科学院、北京协和医学院药物研究所分子免疫与分子药理实验室, 北京 100050) 摘要: 本文探讨 TLR2 是否介导博莱霉素 (BLM) 诱导的急性肺损伤、炎症与纤维化的发生。流式细胞术用于检测树突状细胞 (DCs) 成熟; ELISA 方法检测细胞因子分泌; Western blotting 检测蛋白表达与活化。小鼠气管内注入 BLM 制备急性肺损伤模型。结果表明, 模型小鼠肺局部高表达 TLR2 (P < 0.001) 及其相关信号分子。阻断 TLR2 显著抑制 BLM 诱导的 DCs 成熟及细胞因子 IL-6 (P < 0.001) , IL-17 (P < 0.05) 与 IL-23 (P < 0.05) 的分泌。阻断 TLR2 不仅抑制支气管肺泡灌洗液中炎性细胞的增加, 而且还增强 TH1 (P < 0.05), 抑制 TH2 (P < 0.001)、 Treg (P < 0.01) 与 TH17 (P < 0.01) 反应。重要的是, 阻断 TLR2 可显著减轻肺损伤、炎症与纤维化, 提高动物的生存率 (从 50%到 92%, P < 0.01)。结果提示, TLR2 可作为治疗急性肺损伤与肺纤维化的潜在药物靶点。关键词: TLR2; 博莱霉素; 树突状细胞; 肺部炎症; 急性肺损伤; 纤维化 Received 2010-01-08. Project supported by grants from the National Major Basic Research Program of China (973: #2006CB503808) and from the National Natural Science Foundation of China (30672468; 30901814). * Corresponding author Tel: 86-10-83165034, Fax: 86-10-83161187, E-mail: [email protected]

LIU Han-zhi, et al: Toll like receptor 2 mediates bleomycin-induced acute lung injury, inflammation and fibrosis in mice · 977 ·

Acute lung injury (ALI) may be resulted from various insults or injuries such as infection, oxidative stress, radiation, and chemotherapeutic agents[1]. Acute inflammation and fibrosis are major consequence of ALI and often manifested in multiple lung diseases, such as acute respiratory distress syndrome (ARDS), acute interstitial pneumonia, and idiopathic pulmonary fibrosis[2, 3]. Bleomycin (BLM) has been used in clinics for the treatment of multiple types of cancer, including testicular cancer, lymphoma, and squamous cell cancer in the head, neck, lung, cervix, and other sites. It is estimated that 3%–5% of patients treated with BLM develop dose-dependent interstitial pulmonary fibrosis[4]. Endotracheal challenge in mice with BLM represents a well established model of ALI resulting in pulmonary fibrosis that resembles idiopathic pulmonary fibrosis[5]. Although the pathogenesis of ALI and pulmonary fibrosis is not completely understood, accumulating evidence suggests immune responses play a critical role in the development of lung injury and pulmonary fibrosis. The TH1 response contributes to pulmonary inflammation, injury and resolution, whereas TH2/Treg responses contribute to the protection of lungs from inflammatory injury and to the accumulation of the extracellular matrix, which lead to idiopathic pulmonary fibrosis[1]. Pattern recognition receptors, especially TLRs, play pivotal roles in initiating inflammation, which contributes to the pathogenesis of various lung diseases[6]. The activation of TLRs can determine the polarization of innate and adaptive immune response using DCintrinsic and extrinsic mechanisms[7]. Immature DCs in peripheral tissues undergo dynamic and coordinated reprogramming of gene expression, phenotype and function when TLRs interact with certain molecular motifs, including the exogenous pathogen-associated molecular pattern (PAMP) or the endogenous damageassociated molecular pattern (DAMP) molecules[8]. In addition, TLRs play a critical role in the initiation of innate and adaptive immune responses by recognizing PAMP molecules (such as certain chemotherapeutic drugs) or DAMP molecules (such as endogenous molecules released by cellular injury)[9]. Indeed, TLR2 activation is a critical event that triggers NF-κB activation and the secretion of cytokines and chemokines during BLM exposure[10]. Thus, we hypothesize that TLR2 can mediate BLM-induced ALI, inflammatory responses, and pulmonary fibrosis. Our results

demonstrate that TLR2 activation is an early critical event for the promotion of DC maturation as well as cytokine production by BLM. Importantly, blocking TLR2 signaling significantly attenuates BLM-induced injury, inflammation and fibrosis, and subsequently decreases animal death, suggesting that blocking TLR2 is an effective strategy for the treatment of lung injury and fibrosis via modulation of immune microenvironment in injured lung.

Materials and methods Reagents Ultra pure Ec-LPS (purified from Escherichia coli 0111: B4 strain, TLR4 ligand), Pam3Cys (TLR2 ligand) and mouse recombinant (mr) GM-CSF were obtained from InvivoGen (San Diego, CA). FITC-, PE-, PE-cy5- or APC-conjugated anti-CD11c, MHC II, CD86, CD80, CD40, TLR2, TLR4, CD4 and CD25, FoxP3, IL-4, IL-17, IL-23, IFN-γ and PDCA-1 monoclonal antibodies (mAbs) were purchased from eBioscience (San Diego, CA). Neutralizing mTLR2 Ab (anti-TLR2 Ab) and mTGF-β1 Ab were from R&D System Inc (Minneapolis, MN). Neutralizing mTLR4 Ab (anti-TLR4 Ab) was from Biolegend (San Diego, CA). Anti-ERK, p38, NF-κB p65, I-κB, AP-1, phospho-ERK and phospho-I-κB Abs were from Santa Cruz Biotechnology Inc (San Diego, CA). Phospho-p38 (Thr180/182) Ab was purchased from Cell-Signaling Technology Inc (Massachusetts, MN). BLM (1 u·mg−1) was from Nippon Kayaku (Tokyo, Japan). ECL and ECL plus Western blotting detection reagents were from Amersham Biosciences (Piscataway, NJ). The ELISA kits for MCP-1, IL-6, IL-12, IL-17, IL-23, TGF-β1 and IL-10 were from eBioscience (San Diego, CA). The endotoxin levels in the solutions of BLM and neutralizing Ab were < 0.01 ng·mL−1, as determined by the Limulus amebocyte lysate assay (BioWhittaker; Walkersville, MD). Culture and maintenance of DCs Mouse precursor DCs (CRL-11904 ATCC) were maintained in α-MEM supplemented with 20% fetal bovine serum (FBS, BioWhittaker, MD), 1 mmol·L−1 sodium pyruvate, 0.1 mmol·L−1 nonessential amino acids, 0.05 mmol·L−1 2-mercaptoethanol, 100 u·mL−1 penicillin and streptomycin, and 5 ng·mL−1 mrGM-CSF (Peprotech, Rock Hill, NJ). To evaluate the regulatory effects of BLM on the phenotype and activity of DCs, immature DCs were cultured in 6-well plates at a density of 5×105 cell·mL−1 and BLM at the indicated doses was added.

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To investigate the role of TLR2 or TLR4 in BLMinduced DC maturation, anti-TLR2, anti-TLR4 or isotype-matched Ab (10 µg·mL−1) was added one hour before BLM addition. The neutralizing activities of anti-TLR2 and anti-TLR4 Ab were previously demonstrated[11, 12]. Flow cytometry of DCs Surface markers of DCs were analyzed using flow cytometry as described previously[13]. In brief, DCs either untreated or stimulated with BLM for 24 h were harvested, washed, and suspended in cold 0.01 mol·L−1 PBS (pH 7.4) containing 3% FBS and 0.02% NaN3, and then serially incubated with saturated concentrations of FITC-, PE- and/or PE-cy5-conjugated mAb for 1 hour at 4 ℃. Isotypematched mAb was used in control samples. After incubation, at least twenty thousand stained cells were analyzed using CellQuest software (Becton Dickinson). Quantitative PCR Total RNA was prepared using Trizol reagent following the manufacturer’s instructions (Invitrogen Corp.), and cDNAs were reverse transcribed from 1.0 μg of total RNA. Each 25 µL real time PCR reaction consisted of 5 µL of cDNA, 12.5 µL of 2×SYBR Green PCR Master Mix (Applied Biosystems) and 3.75 µL of 50 nmol·L−1 sense and anti-sense primers. Primer sequences were designed using Primer Express Software (PerkinElmer Life Sciences) and are presented in Table 1. Quantitative PCR was performed on ABI 7000 PCR Instrument (PerkinElmer Life Sciences) using 3-stage program parameters as follows: 2 min at 50 ℃, 10 min at 95 ℃, and then 40 cycles of 15 s at 95 ℃, 30 s at 60 ℃ and 30 s at 72 ℃ , and eventually, 10 min at 72 ℃ . Specificity of the amplification products was confirmed by examination of dissociation reaction plots. A distinct single peak indicated that a single DNA sequence was amplified during PCR. In addition, end reaction products were visualized on ethidium bromidestained 1.4% agarose gels. The appearance of a single band of the correct size confirmed the specificity of the PCR. The results were analyzed by ABI PRISM 7000 SDS software (version 1.0, Applied Biosystems). Each sample was tested in triplicate with quantitative PCR, and samples obtained from at least three independent experiments were used to calculate the mean and standard deviation (SD). Values were expressed as arbitrary units (AU) relative to β-actin. Western blotting analysis Cytoplasmic and nuclear fractions were prepared as described previously[14].

Table 1

Primers for real time RT-PCR

Gene of interest β-Actin

Sense

Primer sequence 5'-CAGCTTCTTTGCAGCTCCTTCGTT-3'

Anti-sense 5'-TACTCCTGCTTGCTGATCCACATC-3' MyD88

Sense

5’-CCCAATGATATCGAGTTTGT-3’

Anti-sense 5’-TTCTTCATCGCCTTGTATTT-3’ MD-2

Sense

5’-ACGCTGCTTTCTCCCATATT-3’

Anti-sense 5’-CATTGGTTCCCCTCAGTCTT-3’ TIRAP

Sense

5’-TGTGTGGCTGCCCTATTTTA-3’

Anti-sense 5’-GAAAACACAGCATGGACCAC-3’ TRIF

Sense

IFN-γ

Sense

5’-AGGCAGGTTTCTTGTGTAAA-3’

Anti-sense 5’-ATGTGCAACTGGGTATTCTC-3’ 5’-CATCAGCAACAACATAAGCGTCA-3’

Anti-sense 5’-CTCCTTTTCCGCTTCCTGA-3’ IL-6

Sense

5’-CTGCAAGAGACTTCCATCCAGTT-3’

Anti-sense 5’-GAAGTAGGGAAGGCCGTGG-3’ IL-10

Sense

5’-TGTGAAAATAAGAGCAAGGCAGTG-3’

Anti-sense 5’-CATTCATGGCCTTGTAGACACC-3’ IL-17

Sense

5’-AGGCCCTCAGACTACCTCAACC-3’

Anti-sense 5’-GCCTCTGAATCCACATTCCTTG-3’ IL-23 p19

Sense

5’-AAAATAATGTGCCCCGTATCCAG-3’

Anti-sense 5’-GCTCCCCTTTGAAGATGTCAG-3’ TGF-β1

Sense

5’-ATCCTGTCCAAACTAAGGCTCG-3’

Anti-sense 5’-ACCTCTTTAGCATAGTAGTCCGC-3’ CXCL5

Sense

5’-CTCAGTCATAGCCGCAACCGAGC-3’

Anti-sense 5’-CGCTTCTTTCCACTGCGAGTGC-3’ CXCL9

Sense

5’-TGTGGAGTTCGAGGAACCCT-3’

Anti-sense 5’-TGCCTTGGCTGGTGCTG-3’ Procol I

Sense

5’-TTCACCTACAGCACGCTTGTG-3’

Anti-sense 5’-GATGACTGTCTTGCCCCAAGTT-3’

Protein concentrations were determined with Coomassie Plus reagent. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were then incubated overnight with primary antibodies. The membranes were incubated with appropriate HRP-conjugated Ab followed by the detection of signal with an enhanced chemiluminescence detection system (Amersham Biosciences). Induction of lung injury and pulmonary fibrosis by BLM Male C57BL/6 mice (6−8 weeks old, SPF grade, No. 0089895) were obtained from Vital River Lab Animal Technology, Co., Ltd (Beijing, China). All animals were housed in pathogen-free and isolated ventilated cages, and were given standard laboratory chow and tap water ad libitum. After measurement of their body weight, animals were anesthetized with an intraperitoneal injection of 50 mg·kg−1 pentobarbital sodium (Merck Sharp Dohme Ltd, Woodbridge, NJ). BLM was dissolved in sterile 0.01 mol·L−1 PBS (pH 7.4) and instilled intratracheally at a dose of 3 u·kg−1


(body weight) on day 0 as previously described[15]. Sham groups received an identical volume of sterile 0.01 mol·L−1 PBS (pH 7.4). Mice (n ≥ 15) were randomly assigned to each of the indicated treatment groups. Anti-TLR2, anti-TLR4 Ab or isotype-matched rat IgG (2 μg/mouse) in saline 200 μL was injected intravenously (iv) one day before BLM administration and again on day 3, 8 and 15 after instillation. Mice were then sacrificed by excessive anesthesia for the collection of bronchoalveolar lavage fluid (BALF) and lungs on day 7 and 28 after BLM instillation. Lung tissue samples were excised and fixed for morphological evaluation or frozen in liquid nitrogen for the measurement of hydroxyproline content. BALF collection and analysis BALF was collected and harvested as previously described[16]. 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 0.9−1.0 mL of BALF was retrieved per mouse, placed on ice, and centrifuged at 500×g for 5 min at 4 ℃. Supernatants were decanted and frozen at −80 ℃ for subsequent use. The cell pellet was resuspended and washed twice in Hank’s balanced salt solution. The cellular viabilities were greater than 99% as assessed by 0.4% trypan blue exclusion. The cells were counted using a hemocytometer to determine alterations in eosinophils, neutrophils, monocytes, lymphocytes and white blood cells, or analyzed by flow cytometry to determine the alterations of CD11c+ mDCs, PDCA-1+ pDCs, CD4+ IFN-γ+ TH1 cells, CD4+ IL-13+ TH2 cells, CD4+ IL-17+ TH17 cells and FoxP3+ CD4+ CD25+ Tregs. ELISA analysis Commercially available ELISA kits (R&D Systems, Minneapolis, MN) were used to measure MCP-1, IL-6, IL-10, IL-12p70, IL-17, IL-23 and TGF-β1 secreted by DCs according to the manufacturer’s instructions. Histopathological assessment of pulmonary inflammation, injury and fibrosis Lungs were excised at day 7 or 28 after the treatment with BLM or PBS, stained with Hematoxylin and Eosin to detect inflammation and injury, and with Masson’s Trichrome to detect collagen. To quantify lung inflammation and injury, ten randomly chosen fields of the left middle lobe at a magnification of ×100 were semi-quantitatively scored as previously described[17]. In brief, the following parameters were analyzed: intra-alveolar or interstitial

inflammation, edema, endothelialitis, bronchitis, pleuritis, and thrombi formation. Two investigators scored all sections in a blinded manner. The total scores of lung inflammation were expressed as the sum of the scores for each parameter. The average collagen deposition was determined by the integrated optical density (IOD) using Image-Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD) in 10 random regions per tissue sample at a magnification of 200×. Measurement of hydroxyproline content To determine collagen deposition in lungs, total hydroxyproline content in lungs was measured by a revised Reddy GK’s method[18]. In brief, lungs were hydrolyzed with 2.5 mol·L−1 NaOH at 120 ℃ , 0.1 kPa for 40 min. The hydrolyzates were diluted with distilled water after neutralization with hydrochloric acid. The content of hydroxyproline in hydrolyzates was measured calorimetrically at 550 nm with pdimethylamino-benzaldehyde. The results were represented as μg per lung. Statistics For the in vitro results, data were expressed as the mean ± standard deviation (SD). For the in vivo results, data were expressed as the mean ± standard error (SE). Statistical differences between groups were compared by analysis of variance (ANOVA) followed by a Dunnett’s multiple comparisons test with P < 0.05 considered statistically significant. Unless specifically noted, analysis was performed with n = 6 − 8 per group in a given assay. The survival rates were analyzed by the Kaplan-Meier method.

Results 1

BLM induced the activation of TLR2 To investigate the regulatory effects of BLM on the expression and activation of TLR2 and TLR4 in lungs of mice in vivo, the expression of TLR2 and TLR4 in lungs was analyzed by flow cytometry, and the expression of TLR adaptors was determined by real-time PCR. The infiltration of TLR2+ and TLR4+ cells was significantly enhanced in lungs of BLMtreated mice (Figure 1A-B). In addition, the expression of MyD88 (P < 0.001), TIRAP (P < 0.01) and TRIF (P < 0.05) was markedly upregulated in lungs of BLMtreated mice (Figure 1C-E), but the expression of MD-2 was not (Figure 1F). This indicated that the TLR2 signaling pathway was fully activated, but the TLR4 signaling pathway was partially activated in BLMinduced injured and fibrotic lungs.

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Figure 1 BLM induced the activation of TLR2 and TLR4. BLM (3 u·kg−1) was administered intratracheally to male C57BL/6 mice. The mice were sacrificed with pentobarbital after 28 days of BLM treatment. By flow cytometry, it was found that the infiltration of TLR2+ (A) and TLR4+ (B) cells was enhanced in the lungs of BLM-treated mice. Results are presented as the mean percentage ± SD of at least three independent experiments. The expression of MyD88 (C), TIRAP (D) and TRIF (E) was upregulated in the lungs of BLM- treated mice, but MD-2 (F) was not. The mRNA expression levels of MyD88, TIRAP, TRIF and MD-2 in the lungs of mice were determined by quantitative PCR. The quantitative results are illustrated as mean relative mRNA expression from 3 reduplicative measurements (shown as fold of sham) ± SD. * P < 0.05, **P < 0.01, ***P < 0.001 vs sham group

2 TLR2 mediated BLM-induced maturation of DCs and alteration of pro-inflammatory cytokine secretion of DCs To investigate whether TLR2 was directl y activated by BLM, anti-TLR2 and anti-TLR4 Ab were used to determine if TLR2 or TLR4 was involved in BLM-induced DCs maturation and cytokine production. The blocking activity of anti-TLR2 and anti-TLR4 Ab was demonstrated previously. BLM significantly promoted the maturation of DCs, which was represented as the increase in the number of CD11c+ MHC I+ DCs, CD11c+ MHC II+ DCs (Figure 2A-B), CD40+CD80+ DCs and CD40+CD86+ DCs (Figure 2C-D). Anti-TLR2, but not anti-TLR4 or isotype-matched Ab, significantly blocked BLM-increased CD11c + MHC I+ DCs and CD11c + MHC II + DCs (Figure 2A-B). Moreover, anti-TLR2 Ab significantly inhibited the expression of CD40, CD80 and CD86 (Figure 2C-D). Blocking TLR2 significantly attenuated BLM-induced production of IL-6 (P < 0.001), IL-17 (P < 0.05), IL-23 (P < 0.05) and TGF-β1 (P < 0.01) (Table 2). Blocking TLR2 also significantly enhanced BLM-induced release of IL-12p70 (P < 0.05), but did not affect the production of IL-10 (Table 2). However, blocking TLR4 enhanced the production of IL-17 (P < 0.05) and TGF-β1 (P < 0.05), but inhibited the production of IL-12p70 (P < 0.05, Table 2). These results indicate that TLR2, but not

Figure 2 TLR2 mediated BLM-induced maturation of DCs. Immature DCs were treated with anti-TLR2, anti-TLR4 or isotype−1 −1 matched Ab (10 μg·mL ) one hour before BLM stimulation (20 mu·mL ). After culturing for 24 h, the cells were collected, washed and stained with respective fluorescence-labeled Ab. The expressions of surface molecules, including CD11c and MHC I (A), CD11c and MHC II (B), CD40 and CD80 (C), CD40 and CD86 (D) were analyzed using flow cytometry. Representative dotplot images from three independent experiments with identical results are shown

LIU Han-zhi, et al: Toll like receptor 2 mediates bleomycin-induced acute lung injury, inflammation and fibrosis in mice · 981 · Table 2 TLR2 mediated the secretion of pro-inflammatory cytokines in DCs induced by BLM. Anti-TLR2, anti-TLR4 or isotypematched Ab (10 μg·mL−1) was added to the DCs one hour before BLM stimulation (20 mu·mL−1). The levels of IL-6, IL-12, IL-17, IL-23, TGF-β1 and IL-10 were determined by ELISA. Results are presented as mean±SD of at least three independent experiments. ## P < 0.01, ### P < 0.001 vs medium group; * P < 0.05, ** P < 0.01, *** P < 0.001 vs BLM group Group

IL-6/pg·mL −1

IL-12/pg·mL −1

IL-17/pg·mL −1

IL-23/ng·mL −1

TGF-β1/pg·mL −1

IL-10/pg·mL −1

Medium

175.56 ± 5.04

16.76 ± 0.72

101.39 ± 31.06

1.23 ± 0.32

116.90 ± 8.82

4.70 ± 0.07

BLM

427.14 ± 4.38###

24.84 ± 0.74##

218.48 ± 20.71##

3.50 ± 0.65##

270.55 ± 25.36##

6.41 ± 0.17

BLM+anti-TLR2

200.34 ± 9.67 ***

36.54 ± 0.79 *

130.67 ± 13.21 *

1.78 ± 0.19 *

109.54 ± 20.58 **

6.43 ± 0.32

*

*

2.64 ± 0.26

367.62 ± 13.60 *

6.65 ± 0.36

3.54 ± 0.28##

253.64 ± 28.31##

6.74 ± 0.25

BLM+anti-TLR4

396.90 ± 23.52

16.02 ± 0.38

BLM+IgG

398.74 ± 10.08###

32.91 ± 0.43###

263.47 ± 24.28

198.14 ± 22.49##

TLR4 mediates the immuno-regulatory effects of BLM. 3 TLR2 mediated BLM-stimulated expression of TLR2 and TLR4 via the activation of p38 kinase and ERK signal pathways To gain insights into the potential intracellular signaling mechanisms that mediate BLM-induced DC responses, two major MAP-kinase signaling pathways, p38 and ERKs were focused on. TLR4 agonist E.coli

LPS significantly induced the phosphorylation of p38, but TLR2 agonist Pam3Cys did not (Figure 3A), which was consistent with previous reports[19]. BLM exposure also induced the phosphorylation of p38 MAPK in DCs (P < 0.05, Figure 3A). Blockade of TLR2, but not TLR4, resulted in significant inhibition of p38 phosphorylation (P < 0.05, Figure 3A). Additionally, BLM significantly enhanced the expression of NF-κB

Figure 3 BLM significantly activated p38-NF-κB and ERK-AP-1 signaling pathways via activation of TLR2 in DCs. Immature DCs were pretreated with or without anti-TLR2, anti-TLR4 or isotype-matched Ab (10 µg·mL−1) for 30 min before BLM (20 mu·mL−1) treatment. Eight hours after BLM treatment, phosphorylation of the specific proteins was detected by Western blotting analysis using antibodies to total- or phosphorylated-p38 (A), NF-κB (B), I-κB, phosphorylated-I-κB (C), ERK (D and E), phosphorylated ERK (E) and AP-1 (F). To normalize the phosphorylation levels of p38, ERK and I-κB to total amounts of p38, ERK and I-κB protein, membranes were stripped and re-probed with an antibody that recognized both phosphorylated and non-phosphorylated forms of p38, ERK and I-κB. Representative results from 5 experiments are shown. Results were presented as mean folds of control ± SD of 5 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 vs control group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs BLM group. Cont: Control

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(P < 0.05, Figure 3B) and inhibited the expression and phosphorylation of I-κB (P < 0.001, Figure 3C). Importantly, BLM-induced activation of NF-κB and degradation of I-κB were blocked by anti-TLR2 Ab in DCs (Figure 3B-C). ERKs are the focal point for diverse cell growth and proliferation, and the critical factor required for the production of TH2/Treg cytokines. Activation of ERK1/2 induces the transcription of the AP-1 gene. BLM treatment significantly enhanced the expression of ERK1/2 in a time dependent manner (Figure 3D). The presence of BLM also markedly promoted ERK1/2 phosphorylation and translocation of AP-1 (P < 0.001, Figure 3E-F). Moreover, anti-TLR2 Ab blocked the BLM-induced increase in the activity of ERK1/2 and translocation of AP-1 (Figure 3E-F). Pam3Cys induced significant phosphorylation of ERK1/2 and translocation of AP-1 (Figure 3E-F), which was consistent with a previous study[20]. 4 TLR2 mediated BLM-induced ALI, inflammation and fibrosis To determine if TLR2 mediates BLM-induced ALI, inflammation and fibrosis, BLM was intratracheally administered, and then animal death, pulmonary injury, inflammation and fibrosis were analyzed. Instillation of BLM (3 u·kg−1) resulted in a 50% death rate of animals (Figure 4A) and significantly increased ALI, pulmonary inflammation and fibrosis (Figure 4B-F).

Administration of anti-TLR2 Ab, but not isotype-matched Ab one day prior to BLM instillation, significantly reduced BLM-induced ALI (Figure 4B), pulmonary inflammation (Figure 4C-D), deposition of collagen (Figure 4C and E), and the content of hydroxyproline (Figure 4F), and subsequently markedly decreased BLMinduced animal death (Figure 4A). It was shown previously that multiple inflammatory cells, including eosinophils, neutrophils, monocytes, lymphocytes, DCs, TH1 cells, TH2 cells, and Tregs etc, possessed distinct roles in BLM-induced lung injury and fibrosis[21, 22]. Indeed, BLM administration significantly increased the counts of these inflammatory cells in BALFs, including eosinophils, neutrophils, monocytes, lymphocytes and white blood cells, not only during the acute stage (day 1 to day 7 after BLM administration), but also during the chronic stage (day 8 to day 28 after BLM administration) (Figure 5A-E). Importantly, anti-TLR2 Ab reduced the BLM-induced increase of these cells (Figure 5A-E). Similarly, targeting TLR2 markedly reduced BLMstimulated MCP-1 production in BALFs on both day 7 and 28 after BLM administration (P < 0.05, Figure 5F). Furthermore, BLM significantly increased the infiltration of CD11c+ mDCs (P < 0.05), PDCA-1+ pDCs (P < 0.05), CD4+ IFN-γ+ TH1 cells (P < 0.01), CD4+ IL-17+ TH17 cells (P < 0.01), and FoxP3+CD4+CD25+ Tregs (P < 0.01, Figure 6A-C, E and F), but not CD4+ IL-13+ TH2 cells

Figure 4 Blocking TLR2 protected BLM-treated mice from ALI, inflammation and fibrosis. BLM (3 u·kg−1) was administered intratracheally to male C57BL/6 mice. Anti-TLR2 or isotype-matched Ab was administered iv one day before BLM administration and again on day 3, 8 and 15 after instillation. Mice were sacrificed on day 7 for the determination of ALI (B) or on day 28 for the examination of pulmonary inflammation, injury and fibrosis (C-F). Targeting TLR2 significantly increased the cumulative survival rates of BLM-administered mice (A). Blockade of TLR2 markedly inhibited BLM-induced acute pulmonary injury and lung inflammation (B). Left, HE staining section; right, summary of inflammation score. Blockade of TLR2 markedly attenuated pulmonary inflammation, injury and fibrosis, which are represented as the representative Masson staining and HE staining sections (C), the summary of inflammation score (D), collagen deposition (E) and hydroxyproline content (F). Data are presented as mean ± SE (n = 15) from two separate experiments. # P < 0.05, ## P < 0.01 vs sham group; * P < 0.05, ** P < 0.01 vs BLM group


Figure 5 Blockade of TLR2 decreased BLM-stimulated recruitment of inflammatory cells in BALFs. Drugs were administered as described in the legend of Figure 4 and mice were sacrificed with excess pentobarbital. The BALF of mice was obtained to analyze the counts of inflammatory cells, including eosinophils (EOS, A), neutrophils (NEUT, B), monocytes (MON, C), lymphocytes (LYM, D) and white blood cells (WBC, E) by a hemocytometer. The level of MCP-1 in BALF was measured using the ELISA method (F). Data are represented as mean ± SE (n = 12). # P < 0.05, ## P < 0.01, ###P < 0.001 vs sham mice on day 7 after BLM treatment; * P < 0.05 vs BLM-treated mice on day 7 after BLM treatment; $ P < 0.05, $$P < 0.01 vs sham mice on day 28 after BLM treatment; ^P < 0.05, ^^ P < 0.01 vs BLM-treated mice on day 28 after BLM treatment

Figure 6 Blockade of TLR2 markedly affected the counts of multiple immune cells in BALF, including CD11c+ mDCs (A), PDCA-1+ pDCs (B), CD4+ IFN-γ + TH1 cells (C), CD4+ IL-13 + TH2 cells (D), CD4+ IL-17+ TH17 cells (E) and FoxP3+ Tregs (F). Mice were treated with drugs as described in the legend of Figure 4 and sacrificed with excess pentobarbital. The BALF of mice were obtained to analyze the counts of these immune cells using flow cytometry. Data are presented as mean ± SE (n = 15) from three separate experiments. # P < 0.05, ## P < 0.01 vs sham mice on day 7 after BLM treatment; * P < 0.05, ** P < 0.01 vs BLM-treated mice on day 7 after BLM treatment; $P < 0.05, $$P < 0.01, $$$ P < 0.001 vs sham mice on day 28 after BLM treatment; ^ P < 0.05, ^^ P < 0.01, ^^^P < 0.001 vs BLM-treated mice on day 28 after BLM treatment

(Figure 6D) on day 7 after BLM treatment. On day 28, BLM stimulation markedly increased the counts of pDCs (P < 0.05), TH2 cells (P < 0.001), TH17 cells (P < 0.01), and FoxP3+ Tregs (P < 0.001) (Figure 6B, D-F), but inhibited the counts of mDCs (P < 0.05) and

TH1 cells (P < 0.05) (Figure 6A and C). TLR2 significantly reversed the regulatory BLM on the infiltration of these immune the production of MCP-1 either on day 7 or after BLM treatment (Figure 6A-F).

Blocking effects of cells and on day 28

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Various cytokines and chemokines are involved in the progression of ALI and pulmonary fibrosis[1]. During the acute stage of lung inflammation, blockade of TLR2, significantly inhibited the expression of IL-6 (P < 0.01), IL-10 (P < 0.05), IL-17 (P < 0.05), IL-23 (P < 0.01), TGF-β1 (P < 0.01), and CXCL5 (P < 0.01) (Figure 7B-G). Moreover, it enhanced the expression of IFN-α (P < 0.01) and CXCL9 (P < 0.01, Figure 7A and H), but did not affect the expression of procollagen I (Figure 7I). During the chronic stage of lung inflammation, blockade of TLR2, significantly inhibited the expression of IL-6 (P < 0.05), IL-10 (P < 0.01), IL-17 (P < 0.01), IL-23 (P < 0.01), TGF-β1 (P < 0.05) and procollagen I (P < 0.05, Figure 7B-F, I). However, it enhanced the expression of CXCL9 (P < 0.01, Figure 7H), but did not affect the expression of IFN-α and CXCL5 (Figure 7A and G). Collectively, blocking TLR2 attenuated BLM-induced ALI, inflammation and fibrosis, and consequently improved the survival of BLM-instilled mice by altering the pulmonary immune microenvironment.

Discussion Lung injury and pulmonary fibrosis can be induced by various stimuli, including chemical insults, persistent infections, autoimmune reactions, allergic responses and radiation, but the mechanism that underlies this progression remains poorly understood. The present study demonstrates that TLR2 mediates BLM-stimulated maturation of DCs and the secretion of proinflammation cytokines (e.g., IL-6, IL-17 and IL-23) and chemokines (e.g., MCP-1) in vitro, which are involved in both p38NF-κB and ERK-AP-1 signaling pathways. Importantly, blocking TLR2 signaling markedly reduces BLMstimulated pulmonary inflammation, injury and fibrosis in vivo. Consistently, Razonable et al [10] report that TLR2 activation plays a critical role in BLM-triggered NF-κB activation and IL-8 production in macrophages in vitro. Also, it is recently indicated that TLR2 promotes TH2/TH17 responses by abrogating the type I IFN amplification loop[23]. Moreover, our recent study indicates that targeting TLR2 attenuates BLM-induced pulmonary fibrosis by reversing the immuno-suppressive

Figure 7 The regulatory effects of TLR2 inhibition on the expression of cytokines and chemokines in the lungs of mice. Mice were treated as indicated in the legend of Figure 4. The lungs were analyzed using real-time PCR to determine mRNA expression levels of multiple genes, including IFN-α (A), IL-6 (B), IL-10 (C), IL-17 (D), IL-23p19 (E), TGF-β1 (F), CXCL5 (G), CXCL9 (H) and procollagen I (I). Data are presented as mean fold of BLM ± SE (n = 15) from three separate experiments. # P < 0.05, ## P < 0.01 vs sham mice on day 7 after BLM treatment; * P < 0.05, ** P < 0.01 vs BLM-treated mice on day 7 after BLM treatment; $P < 0.05, $$P < 0.01 vs sham mice on day 28 after BLM treatment; ^ P < 0.05, ^^P < 0.01 vs BLM-treated mice on day 28 after BLM treatment


microenvironment[22]. Our results demonstrate that TLR2 mediates not only BLM-upregulated expression of TLR2, MHC I, MHC II and the co-stimulatory molecules CD40, CD80 and CD86, but also the BLM-enhanced production of cytokines and chemokines, which can facilitate the polarization of TH cells[24]. Engagement of murine CD28 through CD80 promotes a TH1 response while engagement through CD86 promotes a TH2/Treg response. Concomitantly, TLR2 mediates the BLMstimulated production of IL-6, IL-12, TGF-β1 and IL-23, which can control the direction of TH cell responses. Indeed, TLR2 is an unique member of the TLR family which can mediate TH1[25]-, TH2[26]-, Treg[27]- and TH17-biased responses. Our study is the first one to report that TLR2 mediates BLM-induced production of IL-23 and IL-17, two critical cytokines for the establishment and maintenance of inflammatory autoimmune diseases[28]. TH17 is a central mediator in inflammatory processes and connects T-cell stimulation with neutrophil mobilization, which greatly contributes to the tissue injury and the failure of inflammation resolution[29]. Consistently, activation of TLR2 by Borrelia burgdorferi or Mycobacterium contributes to the production of IL-23 by BMDCs and IL-17 by T cells[30, 31]. BLM-induced release of IL-6 and TGF-β1 is also involved in the development of the TH17 response. However, further investigations are needed to indicate the role and mechanisms of TH17 in BLM-induced pulmonary inflammation, injury and fibrosis. Taken together, these effects of BLM on DCs, which are mediated by TLR2, imply that TLR2 plays an important role in acute and chronic inflammatory pathology of the lungs. The identification of TLR2 as a critical molecule at the onset of a pro-inflammatory signaling cascade is an important observation and a significant advance. Our studies indicate that the blockade of TLR2 protects animals from BLM-induced ALI, inflammation and fibrosis. Consistently, it is reported that activated TLR2 signaling involves pro-inflammatory responses and cystic fibrosis[32]. However, the interactions between TLR2 and BLM-produced DAMPs (e.g. hyaluronic acid[33], HSPs[34, 35] or HMGB1) may also be involved in the progression of ALI and fibrosis. During the inflammation stage, persistent DAMP release results in ongoing cellular stress to inhibit T and NK effectors and favors TH2 responses, which facilitate excessive tissue repair and tissue fibrosis via recruiting M2 cells,

mast cells, eosinophils, pDCs, myofibroblast, etc[36]. Indeed, the antagonists of HMGB1 play a protective role in BLM-induced ALI and pulmonary fibrosis in mice[37]. Therefore, prevention of interactions between BLM or BLM-induced DAMPs and TLR2 on infiltrated immune cells, including DCs, TH cells and epithelial cells, may play a critical role in the amelioration of the pro-inflammatory environment induced by BLM, which results in increased survival and decreased pulmonary inflammation, injury and fibrosis. Additionally, a blockade of TLR2 incompletely reduces BLM-induced inflammation and ALI, suggesting the existence of other mechanisms for action for BLM, such as direct cytostatic action. Together, these results suggest that TLR2 is a potential target for diseases involved in inflammation and tissue injury, and that a combination therapy of BLM with antagonistic TLR2 Ab or an inhibitor may improve anti-cancer efficacy of BLM and reduce BLM-induced systemic inflammation and tissue injury.

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