WISP1 mediates lung injury following hepatic ischemia reperfusion dependent on TLR4 in mice
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Hepatic ischemia-reperfusion injury (IRI) is a common pathological phenomenon, which causes hepatic injury as well as remote organ injuries such as the lung. Several mediators, such as oxidative stress, Ca2+ overload and neutrophil infiltration, have been implied in the pathogenesis of liver and remote organ injuries following reperfusion. WNT1 inducible signaling pathway protein 1 (WISP1) is an extracellular matrix protein that has been associated with the onset of several malignant diseases. Previous work in our group has demonstrated WISP1 is upregulated and contributes to proinflammatory cascades in hepatic IRI. However, the role of WISP1 in the pathogenesis of lung injury after hepatic IRI still remains unknown.
Male C57BL/6 mice were used to examine the expression and role of WISP1 in the pathogenesis of lung injuries after hepatic IRI and explore its potential mechanisms in mediating lung injuries.
We found WISP1 was upregulated in lung tissues following hepatic IRI. Treatment with anti-WISP1 antibody ameliorated lung injuries with alteration of cytokine profiles. Administration with rWISP1 aggravated lung injuries following hepatic IRI through excessive production of proinflammatory cytokines and inhibition of anti-inflammatory cytokines.
In this study, we concluded that WISP1 contributed to lung injuries following hepatic IRI through TLR4 pathway.
KeywordsHepatic ischemia-reperfusion injury WNT1 inducible signaling pathway protein 1 Inflammatory cascades Toll like receptor
Bronchoalveolar Lavage Fluid
High mobility group box-1 protein
Inducible nitric oxide synthase
Pattern recognition receptors
reactive oxygen species
Toll-like receptor 4
Ventilator-induced lung injury
WNT1 inducible signaling pathway protein 1
Hepatic ischemia-reperfusion injury (IRI) is a pathological phenomenon event when hypoxic liver undergoes oxygenic blood reperfusion. Usually IRI can be divided into two categories, namely warm ischemia which often occurs in trauma, shock, and liver transplantation with temporary blood interruption and cold ischemia which appears during organ preservation before transplantation. Recent studies have implied several pathological mechanisms in the pathogenesis of IRI including production of reactive oxygen species (ROS), synthesis of inducible nitric oxide synthase (iNOS), and secretion of proinflammatory cytokines and chemokines which leads to immune cell (especially neutrophil) recruitment and inflammatory cascades [1, 2, 3]. Excessive production of proinflammatory cytokines in the serum, including TNF-α, IL-1β and IL-6 in the early phase contribute to the local and remote organ damage [4, 5]. Previous studies have confirmed lung injury in the IRI model . However, the exact mechanism of IRI induced remote organ injury still remains unclear.
Pattern recognition receptors (PRRs) have been demonstrated to participate in the pathogenesis of IRI, with one of the most important members being Toll-like receptor 4 (TLR4) . TLR4 can recognize various kinds of endogenous antigens and is activated in IRI in liver and remote organs . The functions of TLR4 on immune cells are more vital than on hepatocytes although the regulating mechanisms may be different [9, 10]. Besides TLR4 signaling, other PRRs, such as TLR2 and TLR9, have been reported to be involved in the pathogenesis of IRI [11, 12, 13].
WNT1 inducible signaling pathway protein 1 (WISP1) is a secreted extracellular matrix (ECM) protein which is ubiquitously expressed in multiple organs, such as lung, liver, kidney, heart and small intestine . WISP1 belongs to the CCN family which contains 6 members, namely CCN1 (cysteine-rich protein 61, Cyr61), CCN2 (connective tissue growth factor, CTGF), CCN3 (nephroblastoma overexpressed gene, NOV), CCN4 (WNT1 inducible signaling pathway protein-1, WISP1), CCN5 (WISP2) and CCN6 (WISP3) . The functions of WISP1 has been linked to cell proliferation, survival and differentiation . Recent studies have demonstrated that WISP1 relative expression is significantly upregulated in some diseases, including lung carcinoma, hepatocellular carcinoma  and colon adenocarcinomas . Interestingly, Li et al. have linked WISP1 to the progression of inflammation , that is, stimulation of recombinant WISP1 aggravates proinflammatory responses in LPS stimulated macrophages. Previous work in our group has demonstrated WISP1 is upregulated and contributes to proinflammatory cascades in hepatic IRI . However, the role of WISP1 in the pathogenesis of lung injury after IRI still remains unknown.
In this study, we aimed to investigate the WISP1 expression in the lung tissue after hepatic IRI and determine the regulating mechanisms and functions of WISP1 in the lung injury after hepatic IRI.
C57BL/6 mice were purchased from Shanghai Laboratory Animal Co Ltd. (SLAC, Shanghai, China). TLR4 knockout (TLR4 KO) mice were kindly provided by Dr. Timothy R. Billiar (University of Pittsburgh, USA). All mice were raised in specific pathogen-free condition. Male mice of 8–10 weeks (weight 18.4.8 ± 1.8 g) old were used for experiments. Animal experiments were authorized by the Ethics Committee of Tongji University.
Induction of hepatic I/R injury model]
The induction of segmental (70%) hepatic hepatic warm I/R injury model was performed as previously described with minor modifications [8, 20, 21]. Mice were treated with isotype control IgG or neutralizing WISP1/WISP2 antibody (MyBioSource, 6 μg/g) intraperitoneally 1 h before ischemia and again at the time of reperfusion. In another experiment, mice were administered with recombinant WISP1 protein (rWISP1, 1 μg/g) or sterile phosphate-buffered saline (PBS, w/o Ca2+/Mg2+) intraperitoneally immediately after reperfusion. Sham mice were sufficiently anesthetized, and then a midline abdominal incision was made. The portal triad was exposed without further treatment for liver ischemia. Mice were sacrificed at the predetermined time points (0 h, 3 h, 6 h, 12 h and 24 h) after reperfusion for collecting serum and lung tissues.
Lung edema measurement
Lung edema was evaluated as previously reported by an increase in the wet-to-dry (W/D) weight ratio of the lungs [21, 22]. The left lung was dissected and weighted before and after drying in a micro oven at 65 °C for 48 h. W/D ratio was then detected.
Alveolar-capillary permeability assay
Evans blue albumin (EBA) was used to measure Alveolar-capillary permeability as previous illustrated . Briefly, the internal jugular vein of mice was injected with EBA (25 mg/kg) 1 h before sacrificed. And then, the right lung and blood samples were available for further steps.
Evaluation of Bronchoalveolar lavage fluid (BALF)
BALF was evaluated as previously publications in our group . Mice were instilled with 1 mL PBS and approximately 80% fluid was retrieved. BALF was kept on ice immediately after recovered and was then centrifuged at 1000×g, 4 °C for 5 min. Supernatants were used for total protein concentration examination and cytokine levels, which can keep at − 80 °C for long-term preservation. Total cell counts were determined using a hemocytometer.
RNA extraction, reverse transcription PCR, Quantative real-time PCR
Total RNA was extracted from lung tissues using TRIzol reagent (Sigma-Aldrich) according to established protocols in our group [21, 24]. The total concentration of RNA was measured at 260 nm with a spectrophotometer (BeckmanCoulter, Brea, CA, USA). First strand complementary DNA (cDNA) synthesis was performed using PrimerScript RT Master Mix (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s protocols. Real-time PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), WISP1, IL-6, TNF-α, IL-10 was performed using SYBR premix Ex Taq™ (Takara) with a Step One Plus real-time PCR system (Applied biosystems, USA), under the manufacturer’s instructions. GAPDH was used as house-keeping gene to normalize the gene expression. Gene relative expression was calculated using the 2-ΔΔCt method. All of the primers were synthesized by Sangon Biotech (Shanghai, China). Primers sequence was as follows: mWISP1 forward 5′- CGCCCGAGGTACGCAATAG, reverse 5′- GCAGTTGGGTTGGAAGGACT, mIL-6 forward 5′- CTGCAAGAGACTTCCATCCAG, reverse 5′- AGTGGTATAGACAGGTCTGTTGG, mTNF-α forward 5′- CAGGCGGTGCCTATGTCTC, reverse 5′- CGATCACCCCGAAGTTCAGTAG, mIL-10 forward 5′- CTTACTGACTGGCATGAGGATCA, reverse 5′- GCAGCTCTAGGAGCATGTGG.
Enzyme-linked immunosorbent assay (ELISA)
BALF supernatants were analyzed for mIL-6, mTNF-α and mIL-10 cytokines using a sensitive commercial ELISA kit (R&D Systems) according to the manufacturer’s instructions.
Western blot analysis
Western blot analysis was performed as previously described in our studies [21, 24]. Lung tissues were dissected and lysed using iced-cold lysis buffer (Beyotime, catalog no. P0013G). Protein concentration was determined via standard BCA assay. After gel electrophoresis, protein was then transferred to a nitrocellulose membrane and blocked using 5% nonfat milk for 1 h. Membranes were incubated with rabbit anti-mouse WISP1 antibody (1:400, R&D systems) and β-actin antibody (1:10000, Proteintech) at 4 °C overnight, and then incubated with secondary antibody for 1 h at 37 °C. Odyssey image analysis system (Licor Biosciences) was used to quantify.
Immunofluorescent staining was performed using standard protocols [25, 26]. Briefly, 4–6-mm frozen sections from murine lung tissues were incubated with anti-WISP1 antibody (abcom, 1:200) antibody overnight at 4 °C. Sections were then incubated with secondary Alexa Fluor 488-labelled rabbit anti-mouse antibody (CST, 1:200) for 1 h at room temperature. Nucleus was stained with DAPI for 5 min at room temperature. The positive signals were analysed using a confocal fluorescence microscope (Zeiss LSM510 Confocal).
Determination of myeloperoxidase (MPO) level
The lung MPO level was determined using a commercial mouse MPO ELISA kit (Hycult Biotech) according to the manufacturer’s instructions.
Data were displayed as mean ± SEM. Statistical analysis was performed using GraphPad Prism 5 program. Differences between groups were compared using the Student t-test or one-way analysis of variance (ANOVA). Statistically significant differences were considered as P < 0.05.
WISP1 is highly increased in lung tissue following hepatic IRI
Treatment with anti-WISP1 antibody ameliorates lung injury following hepatic IRI
Treatment with anti-WISP1 antibody alters cytokine profiles in lung tissue following hepatic IRI
Administration with rWISP1 aggravates lung injury following hepatic IRI
rWISP1 facilitates Proinflammatory cytokine and inhibits anti-inflammatory cytokine production in lung tissue following hepatic IRI
WISP1 contributes to lung injury in hepatic IRI dependent on TLR4
Hepatic IRI is a common injury followed by liver transplantation, trauma, shock, etc. Besides liver injury, hepatic IRI also induce remote organ injuries, such as lung injury . The underlying mechanisms of hepatic IRI still remain unclear, of which some mediators have been found playing indispensable roles in liver IRI induced lung injury, including high mobility group box-1 protein (HMGB1), adrenaline and N-acetyl-cysteine [28, 29, 30]. In the current study, we reported a new mediator, WISP1, plays a pivotal effect on hepatic IRI induced lung injury.
WNT1 inducible signaling pathway protein 1 (WISP1), one of the most important members in the CCN family, is a secreted extracellular matrix (ECM) which is ubiquitously expressed in various organs and tissues. The molecular function of CCN family has been associated with wound healing, organ fibrosis, cell survival and proliferation [31, 32]. WISP1 has been identified to be involved in the onset of several malignant diseases, such as breast cancer, hepatocellular carcinoma, colon adenocarcinomas, and lung carcinoma, osteoarthritis and lung fibrosis [16, 17, 33, 34]. In addition, we have demonstrated that WISP1 interacts with TLR4 by co-immunoprecipitation and mediates ventilator-induced lung injury (VILI) dependent on TLR4 signaling . We also find WISP1 might contribute to hepatic ischemia reperfusion injury in mice and possibly depends on TLR4/TRIF signaling . However, the role of WISP1 in the lung injury after IRI remains unknown.
This study aims to confirm the role of WISP1 in the lung injury following hepatic IRI and explore the possible regulating mechanisms. Interestingly, we found WISP1 transcript and protein expression were highly increased in the lung tissue following reperfusion for 6 h, 12 h and 24 h compared with sham mice. Immunofluorescence further confirmed WISP1 expression in the cytoplasm of cell in the lung. These results preliminarily indicated that WISP1 not only participated in the liver injury but also was involved in the pathogenesis of lung injury after hepatic IRI. To further validate the role of WISP1 in the lung injury, mice in the sham and IRI group were treated with anti-WISP1 antibody intraperitoneally to neutralize WISP1 in vivo. Fortunately, we found WISP1 neutralization significantly ameliorated lung injury after hepatic IRI compared to control IgG group characterized by less diffuse interstitial edema and inflammatory cell infiltration. Consistently, treatment with anti-WISP1 antibody markedly inhibited TNF-α and IL-6 production and enhanced IL-10 expression, further confirming the ameliorated lung injury. Conversely, administration with rWISP1 aggravated lung injury together with excessive expression of TNF-α and IL-6 as well as inhibition of IL-10 production. Since the expression and function of WISP1 has been closely linked to TLR4 pathway, we next sought to explore the potential relationship between WISP1 and TLR4. Interestingly, we found TLR4−/− mice in IRI group displayed less transcript and protein expression of WISP1 compared with WT mice, preliminarily suggesting that WISP1 expression in lung injury after hepatic IRI was dependent on TLR4. Besides, rWISP1 could not induce lung injury in TLR4−/− mice of IRI group compared with WT mice, further indicating that WISP1-mediated lung injury and inflammatory cascades might also dependent on TLR4. These data suggested there might be a circulatory regulating mechanism between WISP1 and TLR4 in the lung injury following hepatic IRI. However, the specific mechanisms how TLR4 regulates WISP1 expression and how WISP1 mediates inflammatory cascades through TLR4 need to be further elucidated in future studies.
In conclusion, our study demonstrated that WISP1 expression is upregulated in lung tissue following hepatic IRI. Anti-WISP1 antibody ameliorated lung injury with alterations in cytokine profiles. rWISP1 aggravated lung injury through excessive proinflammatory cytokine production and inhibition of anti-inflammatory cytokines. WISP1 contributes to lung injury through TLR4 pathway after hepatic IRI. Medications targeting WISP1 might be a promising approach for patients with lung injury following hepatic IRI.
We thank Prof. Timothy R. Billiar (M.D., Ph.D. Professor, Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA) for providing suggestions of this study and supplying us the TLR4 knockout mice.
We are very grateful for the support from the National Natural Science Foundation of China (No. 81571927 to Li Q).
Availability of data and materials
The dataset of this article are stored in the Laboratory of Shanghai East Hospital and can be made available from the corresponding author upon reasonable request.
QL, YT and ZC designed the study; YT and ZY wrote the manuscript; YT and RZ collected the data; XD, ZY and YT performed the experiments and analyzed the data; ZC helped to review the analysis of data; QL and ZC discussed the results and reviewed the manuscript before submission. All authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
The study was approved by the Ethics Committee of the University of Tongji and the experiments were performed in accordance with the National Institutes of Health Guidelines for the Use of Laboratory Animals.
Consent for publication
All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
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