Sepsis refers to an overwhelming systemic inflammation condition induced by uncontrolled host responses against invading pathogens (1). The high morbidity and mortality of sepsis are mainly due to multiorgan dysfunction, especially acute lung injury/acute respiratory distress syndrome (ALI/ARDS), which is consistent with previous studies reporting that approximately 50% of septic patients developed ALI/ARDS (2, 3). Despite advances in clinical and experimental studies on sepsis, few effective therapies have been developed to improve the outcome of septic ALI/ARDS.
Circulating fluid and inflammatory cells are isolated from tissues by the microvasculature and vascular endothelial cells under normal physiological conditions (4). During septic ALI, pulmonary vascular/microvascular endothelial cells that are central to the integrity of blood–air barrier become the principal targets of harmful mediators, including activated polymorphonuclear neutrophils (PMNs), lipopolysaccharides (LPS), and cytokines (5, 6). Then, hyperpermeability between alveolar and pulmonary microvascular endothelial cells (PMVECs) becomes one of the characteristics of sepsis-induced microvascular dysfunction (7, 8). The presence of alveolar macrophages and migratory PMN-PMVEC adhesion participate in septic PMVEC death (9). These injuries caused extravascular leakage of protein-rich edema into the interstitial and alveolar areas, thus obstructing gas exchange. Our previous study and other studies demonstrated that PMVEC repair and endothelial barrier function maintenance could improve the outcome of sepsis-induced ALI when the homeostasis of pulmonary function was destroyed in septic ALI (10–12).
Fibroblast growth factor-inducible 14 (Fn14) is a cell surface TNF-like weak inducer of apoptosis (TWEAK) receptor that belongs to the TNF family (13). Previous studies reported that the TWEAK/Fn14 pathway was involved in various malignant tumor invasion and inflammation-related pathologies (14, 15). In normal tissues, Fn14 is expressed at relatively low level. However, once injury occurs, Fn14 is overexpressed locally where it exerts specific roles (16, 17). It was also reported that adhesive capacity and inflammatory responses were induced by the TWEAK/Fn14 interaction in human umbilical vein endothelial cells (18). Inhibition of the TWEAK/Fn14 interaction in human cerebral microvascular endothelial cells could attenuate ischemic brain damage by reducing the recruitment of activated inflammatory cells (19). Based on these findings, we hypothesized that the TWEAK/Fn14 pathway may modulate vascular endothelial cell function.
Recently, Masaki et al. reported that serum TWEAK levels were increased higher in septic shock patients and may prove to be a potential biomarker for the assessment of disease severity and mortality in septic patients (20). However, little is known about the role of Fn14 in sepsis, especially septic ALI/ARDS. Therefore, our present study aims to assess the alteration of Fn14 on septic PMVECs, investigate the potential effects of such changes on lung tissues after Fn14 blockade, and explore the underlying mechanisms.
PATIENTS AND METHODS
ALI model establishment
Male C57BL/6 mice (aged 6–8 weeks) were purchased from the Animal Experimentation Center of the Second Military Medical University (Shanghai, China). All experiments were approved by the Institutional Animal Care and Use Committee of Changhai Hospital. The ALI model was induced by CLP surgery under inhaled sevoflurane anesthesia as previously described (10). After opening the peritoneum and exposing the gut, a double puncture of the cecal wall was performed to induce polymicrobial peritonitis. Then, the abdomen was closed in layers. All mice were housed in an air-conditioned room at a constant temperature (24 ± 1°C) and humidity (40%–80%) with a 12 h light–12 h dark cycle. All mice had free access to food and water throughout.
All mice were randomized to four groups: Sham group, CLP group, Isotype group and Anti-Fn14 group. Mice in the Sham group underwent peritoneum opening and gut exposure without ligation and puncture; 3 h after surgery, anti-Fn14 purified monoclonal antibody (ebioscience, Clone: ITEM-4) was injected intraperitoneally (i.p.) at a dose of 100 μg/mouse in 200 μL sterile PBS in the Anti-Fn14 group. An equal volume of IgG was administered i.p. in the Isotype group. Sham or CLP group mice received an equal volume of sterile PBS through i.p. injection. To evaluate the survival rate, mice were monitored for survival every 6 h until 7 days after surgery.
Measurement of Fn14 expression on pulmonary microvascular endothelial cells
All mice were anesthetized with sevoflurane and sacrificed by removing all blood from heart. The left lung tissue was obtained to calculate Fn14 expression on PMVECs 24 h after surgery. First, Fn14 expression in lung tissues was detected by immunohistochemical staining following standard procedures. In addition, left lung single-cell suspensions were prepared as previously described (10, 21). The percentage of Fn14 expression on PMVECs was detected by flow cytometry. Data were obtained by FACSCalibur flow cytometry (BD Biosciences) and analyzed by Flowjo software.
Detection of proteins and activated cells in BALF
As we previously described (10), bronchoalveolar lavage fluid (BALF) was collected by washing the lungs twice with 0.5 mL PBS via the tracheal cannula when mice were sacrificed by CO2 inhalation 24 h after surgery. The protein levels in the supernatants were determined by a BCA Protein Assay Kit (Thermo Fisher Scientific).
Precipitated activated cells were resuspended in PBS were calculated by Flow Cytometry via specific markers (Ly6G for neutrophil, F4/80 for macrophage). The number of each activated cell was calculated by obtaining total cell counts and each subgroup percentage based on FACS analysis.
Analysis of the W/D ratio of lung tissue
All mice were sacrificed by CO2 inhalation 24 h after surgery. Left lung tissues were removed and weighed to obtain the “wet” or “dry’ weight after certain procedures as described previously (10). The wet to dry (W/D) ratio of lung tissue was calculated to evaluate the degree of pulmonary edema.
All mice were sacrificed to obtain the right upper lung lobe for histologic examination without BALF collection. The tissue was fixed in 10% formalin for at least 24 h, paraffin embedded, sliced into 4–5 μm sections, stained with H&E and Masson's trichrome, and scored by two experienced blinded pathologists under a light microscope according to the criteria previously described (10).
According to our study, the lung tissue sections (4–5 μm) were incubated with anti-Fn14 Ab (ebioscience, Clone: ITEM-4), anti-Ly6G Ab (ab25377), anti-CD68 Ab (ab53444), anti-ICAM-1 Ab (ab171123), and anti-MCP-1 Ab (ab8101) at 4°C overnight followed by corresponding secondary antibodies at 37°C for 30 min. All sections were evaluated by two blinded experienced pathologists under a light microscope.
Knock down of Fn14 expression in HPMECs through siRNA transfection.
Human pulmonary microvascular endothelial cells (HPMECs; CRL-3244) obtained from icell bioscience (Shanghai, China) were grown in DMEM with 10% fetal bovine serum (FBS) in a humidified incubator containing in 5% CO2 at 37°C. Cells with 5–8 passages were used for this experiment as previously described (4). Small interfering RNA (siRNA) targeting Fn14 obtained from Genepharma Co, Ltd. (Shanghai, China) was transfected into HPMECs using Lipofectamine 2000. The transfected cells were cultured for an additional 48 h before RNA extraction.
Quantitative real-time PCR
Total RNA was extracted from HPMECs using the TRIzol Reagent (Invitrogen, Mass) according to the manufacturer's instruction. After extraction and quantification of total RNA, the transcriptional level of target genes was quantified by real-time PCR using a standard SYBR Green PCR protocol on a real-time PCR system (Applied Biosystems, Calif). The relative expression levels of the mRNAs mentioned above were normalized to GAPDH expression in each sample. The primers are listed in Supplementary Fig. 1, https://links.lww.com/SHK/A593.
Assessment of endothelial permeability
HPMECs were transfected with siRNA against Fn14 or a negative control. Until 48 h after transfection, the cells were refreshed and stimulated with TWEAK (R&D 1090-TW/CF, 100 ng/mL) for 6 h. According to our previous study (10), transendothelial electrical resistance (TER), which represents cellular barrier properties, was measured by a Millicell ERS-2 voltammeter (Millipore, Billerica, Mass). The resistance value of an empty culture insert (no cells) was subtracted as background.
Left lung tissues or HPMECs (5 × 105 cells) treated with TWEAK for 6 h were homogenized in protein lysis buffer containing protease inhibitor (Gibco, Calif) for 10 min. According to our previous protocol (10), after several processes, the primary antibodies, including Fn14 (ab109365), ICAM-1 (ab53013), MCP-1 (ab151538), and Caspase-3 (ab32351), were incubated with corresponding membranes overnight. And then the membranes were washed with TBS-T and incubated with the secondary antibody for 2 h at room temperature. Finally, the protein bands were visualized using an enhanced chemiluminescence (ECL) western blot kit. The chemiluminescent signals were quantified via densitometry with Image-Pro Plus.
All data were expressed as the mean ± SD. Statistical analysis was performed using SPSS 16.0 Programs (SPSS Inc., Ill). Graphs were plotted by Prism 5.0 (GraphPad Software, San Diego, Calif). The differences between two groups were compared by unpaired Student t test. Multiple-group differences were analyzed by one-way analysis of variance (ANOVA), followed by Tukey multiple comparison test. Survival of the two subgroups was estimated by Kaplan–Meier survival curves; comparisons were performed by the log-rank test. P < 0.05 was considered statistically significant.
Upregulation of Fn14 on pulmonary microvascular endothelial cells in murine sepsis-induced ALI
In this study, we first measured Fn14 expression in lung tissues by immunohistochemistry staining. As noted in the obtained images, Fn14 was overexpressed in the CLP group compared with the Sham group (Fig. 1A), and the ratio of integrated optical density (IOD) in the CLP group was dramatically increased compared with Sham group (Fig. 1C). Furthermore, to calculate the expression of Fn14 on PMVECs, lung single cell suspension was prepared and stained with fluorochrome-conjugated anti-CD31and anti-Fn14 antibodies. As noted in the representative images, PMVECs were gated as the CD31+ subpopulation (Supplementary Fig. 2, https://links.lww.com/SHK/A593). According to the results, the percentage of Fn14 on PMVECs in the CLP group was significantly upregulated compared with the Sham group (58 ± 12.4% vs. 10 ± 2.85%) (Fig. 1, B and D). In addition, we detected changes in Fn14 on other activated cells in peripheral blood (PB) and peritoneal lavage fluids (PLF). Per our results, activated inflammatory cells exhibit low Fn14 expression in the PB, and no statistically significant differences were noted between the Sham and CLP group (Fig. 1E). Interestingly, the percentage of F4/80+ macrophages expressing Fn14 in septic mice PLF was increased compared with the Sham group (Fig. 1E).
Fn14 blockade alleviates the severity of sepsis-induced ALI
To investigate the possible protective role of Fn14 blockade, histological examination, the lung W/D ratio and protein concentrations in the BALF were used to assess the precise protective role of Fn14 blockade in septic ALI. As shown in Fig. 2A, substantial morphological changes, including edema, alveolar collapse, and accumulation of inflammatory cells, were observed in the lung tissues in the CLP and Isotype groups, whereas thickening of the alveolar wall was alleviated. In addition, the infiltration of inflammatory cells was reduced after i.p. injection of Anti-Fn14 mAb. The scores representing lung injury were also significantly reduced (Fig. 2B). Furthermore, Fn14 blockade reduced protein levels in the BALF after CLP surgery (Fig. 2C). Fn14 blockade reduced the W/D ratio reflecting the severity of pulmonary edema (Fig. 2D).
Fn14 blockade inhibits activated inflammatory cell infiltration after septic ALI
Accumulation of activated inflammatory cells in the lungs was directly correlated with the severity of septic ALI. At 24 h post operation, the infiltration of neutrophils and macrophages in lung tissue was assessed by immunohistochemistry.
As shown in Fig. 3, Ly6G+ neutrophils migrated into lung tissues after septic ALI, but Fn14 blockade reduced the number of neutrophils that infiltrated the lungs compared with the CLP group (43.20 ± 9.04 vs.123.40 ± 14.17/HPF). Consistent with this tendency, Fn14 blockade alleviated the counts of Ly6G+ neutrophils in the BALF after CLP surgery (11.84 ± 1.71 vs. 19.95 ± 4.13 × 105/mL). Similar with these findings, Fn14 blockade also decreased macrophage infiltration in lung tissues (41.40 ± 17.40 vs. 159.40 ± 19.48/HPF) and the BALF (6.97 ± 1.75 vs. 13.27 ± 3.96 × 105/mL).
Fn14 knockdown improves the function of HPMECs in vitro
PMVEC dysfunction during sepsis was associated with barrier disability, resulting in edema fluid flowing into organs, especially the lung tissue. In the present study, a stable siRNA (5’-GCUGACACUGACUAA GGAATT-3’ and 3’-UUCCUUAGUCAG UGUCAGCTT-5’) was obtained to investigate the function of Fn14 on HPMECs (Fig. 4, A–C). According to our present study, compared with the negative control (NC) group, Fn14 knockdown in HPMECs induced the upregulation of TER dramatically upon TWEAK stimulation (Fig. 4D). In addition, Fn14 silencing in HPMECs downregulated TWEAK-induced Caspase-3 overexpression (Fig. 4, E and F). These results suggested that apoptosis induced by the TWEAK/Fn14 pathway may partly explain the high-permeability of HPMECs.
Fn14 silencing decreases ICAM-1 and MCP-1 expression in vitro
TWEAK stimulation upregulated ICMA-1 and MCP-1 expression in the NC group, whereas ICAM-1 and MCP-1 expression significantly reduced when Fn14 was silenced (Fig. 4, G–J). These results may partly explain the infiltration of neutrophils and macrophages in lung tissues.
Fn14 blockade reduces ICAM-1 and MCP-1 expression in lung tissues
Similar to our vitro study results, ICAM-1 and MCP-1 were overexpressed in the lung tissues after septic ALI (Fig. 5A), but the IOD values were significantly reduced after Fn14 blockade compared with the CLP group (Fig. 5, D and E). Consistent with the representative immunohistochemical results, ICAM-1 and MCP-1 protein levels in the Anti-Fn14 group were reduced compared with the CLP group when normalized to β-actin (Fig. 5, B and C and F and G).
Fn14 blockade ameliorates sepsis-induced pulmonary fibrosis and improves survival
In this study, we evaluated the long-term protective effect of Fn14 blockade on septic ALI. Collagen deposition in the lung tissues increased significantly after CLP. However, the pulmonary fibrosis scores were reduced at that time point in the anti-Fn14 mAb-treated group (Fig. 6, A and B). Furthermore, all mice in the CLP group died during the 6 days after CLP (Fig. 6C), and the survival rate in the Isotype group was approximately 10% vs. 40% in the Anti-Fn14 group. These exciting results suggest that Fn14 blockade may protect the lung against sepsis-induced injury.
Sepsis-induced ALI/ARDS remains a leading cause of death in ICUs. Increased dysfunction of PMVECs as the main target of insults is associated with pulmonary high-permeability-induced diffuse inflammation, eventually resulting in disruption of gas exchange (4, 7, 22). In this study, we found that targeting the TWEAK/Fn14 pathway by administration of an anti-Fn14 neutralizing antibody or siRNA could produce a protective effect against sepsis-induced ALI.
Given that the serum TWEAK level can be used as an indicator of the severity of sepsis (20), we postulated whether its receptor Fn14 had a similar function. Our results indicated that Fn14 was minimally expressed on activated immune cells isolated from PB before or after CLP surgery (Fig. 1E). Surprisingly, Fn14 expression was dramatically upregulated in lungs after septic ALI (Fig. 1, A and C). In addition, flow cytometry method (FCM) results reinforced our hypothesis that Fn14 expressed on PMVECs was upregulated significantly after CLP surgery (Fig. 1, C and D). These findings suggested that changes of Fn14 expression in PMVECs may play important roles in the pathophysiologic process in sepsis-induced ALI.
Recently, evidences have indicated strong implications of TWEAK/Fn14 signaling in several pathophysiologic processes, including proliferation/apoptosis, angiogenesis, and induction of inflammatory cytokines (23, 24). Inhibiting the TWEAK/Fn14 pathway may improve the outcomes of several diseases, such as lupus nephritis, spinal cord injuries, and cardiac fibrosis (25–27). Interestingly, we found that administration of an anti-Fn14 mAb intraperitoneally ameliorated the parameters that reflect the severity of lung injury, including reducing the pathology, lung W/D ratio, and protein concentration in BALF. Activated cells (neutrophil and macrophage) that infiltrated into septic lung tissues are the main principal factors that participated in the dysfunction of microvascular endothelial cells. Consistent with our previous study (10), the accumulation of activated inflammatory cells that infiltrated into the lungs after CLP was dramatically reduced after neutralizing antibody administration. However, previous studies reported that overexpressed chemokines and adhesion molecules on vascular endothelial cells induced by cascade magnified inflammatory mediators are responsible for the activated inflammatory cells infiltration (17, 18, 28, 29). Previous studies demonstrated that Fn14 was involved in the recruitment of neutrophil via ICAM-1 expression and macrophage infiltration by MCP-1 expression (30, 31). In our study, we evaluated the variation of ICAM-1 and MCP-1 expression when Fn14 was silenced (Supplementary Fig. 3, https://links.lww.com/SHK/A593) or blockade. As expected, Fn14 gene silencing reduced ICAM-1and MCP-1 expression significantly in vivo and in vitro study. These findings may partly explain the variation of neutrophils and macrophages in the lung tissue of septic mice.
However, unlike CD11b+ monocytes in peripheral blood, we unexpectedly found that Fn14 was highly expressed in peritoneal F4/80+ macrophages of septic mice (Fig. 1E), which is similar to the report of Ortiz et al. (32). This finding raised doubts about the protective roles in septic ALI through modulating the function of PMVECs. Therefore, we chose HPMECs to confirm the protective roles of Fn14 silencing in vitro. As expected, enhanced high permeability reflected by lower levels of TER was reversed effectively via Fn14 gene silencing. In addition, Fn14 knockdown reduced the apoptotic ratio induced by TWEAK stimulation based on Caspase-3 levels in HPMECs. As previous studies reported apoptosis is associated with the death of septic PMVECs, eventually resulting in dysfunction of the pulmonary microvascular albumin-permeability barriers (7, 33). These suggested that Fn14 regulates the PMVEC apoptosis and may be independently involved in the maintenance of PMVEC integrity.
In our study, anti-Fn14 mAb administration alleviated the severity of pulmonary fibrosis induced by septic ALI suggesting that Fn14 blockade may ameliorate the long-term detrimental effects of septic ALI. Furthermore, Fn14 knockdown not only modulates the uptake and differentiation in macrophage (34, 35), but also involves in cytokine production and bacterial clearance of macrophages during sepsis in our unpublished study (data not shown). These potentially supporting our result that Fn14 blockade improved the survival of animals with experimental sepsis.
Our study demonstrated that the TWEAK/Fn14 pathway played an important role in septic PMVEC dysfunction. Importantly, this study may provide strong evidence that Fn14 has a significant therapeutic potential as a novel target to overcome septic ALI and supports future efforts in other septic syndromes.
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