Sepsis is a life-threatening multiple organ failure caused by host immune response dysregulation to microbial infection, with high mortality (1, 2). For some reason, pathogens invade blood circulation, multiply (in the blood), and produce many toxins and metabolites, causing severe symptoms. Clinical presentations of sepsis are characterized by the body’s response to microbial infection, including fever, tachycardia, tachypnoea, white blood cell changes, and organ injury distant from the injection site, such as acute lung failure, acute liver injury failure, and acute kidney injury (3). During microbial infection, exogenous pathogen-associated molecular patterns and endogenous damage-associated molecular patterns disrupt most endothelial functions, leading to a procoagulant, antifibrinolytic, and proinflammatory state (4). The structure of the endothelial glycocalyx, a gel-like meshwork of glycosaminoglycans and proteoglycans surrounding the entire luminal surface of the endothelium, is disrupted early during sepsis, resulting in multi-organ failure later (5-7). Heparinase 1 is upregulated in sepsis, and is primarily responsible for glycocalyx degradation (8). Thus, the endothelium is considered a major target for sepsis therapy. However to date, no specific intervention targeting endothelial dysfunction and glycocalyx degradation exists.
Interferon-β (IFN-β) is well known for its anti-viral activity. However, increasing evidence suggests that IFN-β can benefit the host’s defense against bacterial infection (9, 10). We demonstrated that IFN-β protects against lethal endotoxic and septic shock via the upregulation of SIRT1 (11), a nicotinamide adenine dinucleotide-(NAD+)-dependent histone deacetylase that inhibits the immune response via the modulation of p53 and NF-κB functions (12, 13). The study showed that IFN-β increases SIRT1 expression in macrophages, inhibits proinflammatory cytokines, and protects against sepsis. The other group also showed that the intravenous treatment of IFN-β reduced the 28 d mortality by 81% in patients with acute respiratory distress syndrome, a disease with similar underlying mechanisms to sepsis (12). Many patients with sepsis do not have excessive immune activation, but do have immunosuppression (14-17). The SIRT1-mediated inhibition of immune function in macrophages cannot solely explain the protective effect of IFN-β against sepsis. Thus, in this study, we further investigated the role of IFN-β in sepsis, especially that pertaining to endothelial function modulation.
Our previous study showed that the protective effect of IFN-β against sepsis was blocked by dominant negative SIRT1, indicating that SIRT1 is essential in the role of IFN-β. SIRT1 removes acetyl groups from target proteins using NAD+ as its substrate (18). NAD+ directly regulates SIRT1 activity by substrate-dependent activation (19). To enhance IFN-β efficacy by increasing intracellular NAD+, we combined NAD precursors, and tested the effect of the combinations on cecal ligation puncture-(CLP)-induced sepsis in mice. Intravenous administration of IFN-β (1,000 units/20 g) 6 h and 18 h after CLP challenge significantly improved survival rates by 40% on d 10, compared to that in control mice (Fig. 1A-D) (log-rank test, P < 0.01). The combination with NR, but not nicotinamide (NM) and nicotinamide mononucleotide (NMN), enhanced IFN-β-mediated improvement of the sepsis survival rate (Fig. 1A-D). These results suggest that NR is an enhancer in IFN-β-mediated sepsis protection.
To examine the effect of IFN-β plus NR on CLP-induced organ damage, lung, liver, and kidney samples were collected, and a histological examination was conducted. In CLP mice, hematoxylin and eosin (H&E) staining showed the kidney damage score, lung injury score, and liver inflammation score were increased, compared with sham mice (Supplementary Fig. 1A, B). Clinical score changes observed in IFN-β plus NR-treated mice had significantly weaker changes, compared with CLP mice (Supplementary Fig. 1B). The wet/dry weight ratio, an assessment of edema, was significantly increased in CLP mice, compared with sham mice (Supplementary Fig. 1C). IFN-β plus NR treatment significantly reduced CLP-induced increase in the wet/dry weight ratio (Supplementary Fig. 1C). These results suggest that IFN-β plus NR decreases CLP-induced multi-organ damage.
To examine whether endothelial SIRT1 is involved in IFN-β plus NR-mediated sepsis protection, we generated mice with the endothelial cell-(EC)-specific deletion of
We found that lung ECs express SIRT1 (Fig. 1E). To determine the effects of IFN-β on SIRT1 expression, murine yolk sac ECs (MYSECs) were treated with IFN-β in a dose and time course. As shown in Fig. 2, MYSECs were treated with 100, 200, 400, or 1,000 U/ml of IFN-β for 24 h. These treatments resulted in a dose-dependent increase in SIRT1 protein levels (Fig. 2A). Additionally, when MYSECs were treated with 1,000 U/ml of IFN-β for 0, 2, 6, or 24 h, IFN-β significantly increased SIRT1 protein expression in a time-dependent manner (Fig. 2B).
To determine the effects of IFN-β on SIRT1 transcription, MYSECs were treated with IFN-β in a dose and time course as described above, and
We investigated the effect of protein synthesis inhibition on IFN-β-induced increase in SIRT1 protein levels in MYSECs. The inhibition of protein synthesis by cycloheximide (CHX) did not decrease IFN-β-induced increase in SIRT1 protein levels (Fig. 2E), indicating that IFN-β upregulates SIRT1 protein levels by protein stabilization, rather than protein synthesis.
We performed a transwell assay to evaluate the effects of IFN-β on lipopolysaccharide-(LPS)-induced MYSEC permeability. LPS stimulation (300 ng/ml) significantly increased MYSEC permeability by 1.8-fold, compared with vehicle treatment. By contrast, IFN-β plus NR treatment significantly suppressed the hyperpermeability of MYSECs induced by LPS (Fig. 3A). The
Since endothelial glycocalyx is essential to maintaining vascular permeability, we examined the effect of IFN-β plus NR treatment on CLP-induced endothelial glycocalyx damage. We demonstrated that EC-
Seven to eight-week-old male C57BL/6 mice weighing between 20 and 24 g were purchased from Nara-Biotec (Seoul, Korea). The mice were maintained in an environment with a temperature of 22 ± 2°C and a 12-h-light-dark cycle. Food and water were supplied
After mice were anesthetized using 150 mg/kg ketamine and 17.5 mg/kg rompun, a 1-2 cm ventral midline abdominal incision was made on the shaved and disinfected skin of the abdomen. The cecum was then exposed, ligated with 6-0 silk suture just terminal to the ileocecal valve to prevent intestinal obstruction, and punctured through with an 18-gauge needle. The punctured cecum was gently squeezed to extrude a 1-2 mm droplet of feces and then returned to the abdominal cavity. Then, the abdomen was closed, and the mice were intraperitoneally injected with pre-warmed normal saline (2.5 ml/100 g body weight) immediately after the procedure.
Mice were intravenously injected with IFN-β (2 μg/kg) (R&D Systems, Minneapolis, MN, USA), NM (40 μmole/kg) (Sigma-Aldrich), NMN (40 μmole/kg) (Sigma-Aldrich), or NR (40 μmole/kg) (Medkoo Biosciences, Morrisville, NC, USA) twice daily at 6 h and 18 h after CLP. The survival time was recorded daily up to 10 days after CLP or sham surgery.
Seven to eight-week-old WT and EC-
The MYSECs were purchased from ATCC (Manassas, VA, USA) and cultured in 5% CO2 at 37°C in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 0.2% glutamax, and 0.5% penicillin-streptomycin. For the
Tissue and cell lysates (20 μg) were loaded, run on 7.5% sodium dodecyl sulfate-polyacrylamide gels, and transferred to polyvinylidene fluoride membranes as previously described (21). Membranes were blocked with 3% nonfat milk in Tris-buffered saline (pH 7.4) with 0.1% Tween 20 for 1 h at room temperature. The membranes were incubated with primary antibodies against SIRT1 (B-7, sc-74465, 1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA), heparinase 1 (ab228660, 1:500) (Abcam, Cambridge, Cambridgeshire, UK), and β-actin (A5441, 1:5000) (Sigma) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-(HRP)-conjugated secondary antibody for 1 h at room temperature. Protein bands were visualized using an electrochemical luminescence system (Intron Biotechnology, Inc., Seongnam-si, South Korea) and a Fusion Fx7 Spectra (Vilber Lourmat, Collégien, France). Protein expression was quantified using Fusion-Capt software (version 16.08; Vilber Lourmat).
The MYSECs (2.5 × 105 cells/well) were cultured as described above on the top chamber of the transwell inserts (0.4 μm; Corning B.V. Life Sciences, The Netherlands) for 24 h with 300 ng/ml of LPS in the presence of 40 ng/ml of IFN-β and/or 800 μM NR. The media in the upper chambers were exchanged with 300 μl of serum-free media containing 5 μl of streptavidin-HRP (R&D Systems). After 5 min, 20 μl of the media in the lower chambers were transferred to new 96-wells and analyzed for HRP activities by adding 100 μl of 3,3’,5,5’-tetramethylbenzidine substrate (R&D Systems). The absorption at 450 nm was acquired with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
We evaluated vascular leakage using EBD (Sigma-Aldrich) extravasation. Eighteen hours after CLP surgery, 200 μl of 0.5% EBD was administered via the caudal vein. After the dye circulated for 1 h, the organs were excised and imaged. After imaging, the organ tissues were blotted dry, weighed, and homogenized in formamide (Sigma-Aldrich). Following 48-h extraction at 55°C, the tissue fluid was centrifuged at 12,000 ×
For the RT-PCR analysis, the total RNA was extracted from cells or tissues using RNeasy Plus Mini Kit (Qiagen, Dusseldorf, Germany). High-fidelity cDNA was generated from each RNA sample using the Superscript III cDNA amplification system (Invitrogen). Quantitative RT-PCR samples were prepared as a mixture using the Quantitect SYBR Green PCR kit (Takara Bio, Shiga, Japan) following the manufacturer’s instructions. The reaction was performed at 65°C for 5 min, followed by 42°C for 1 h and 95°C for 5 min using a MyCyclerTM Thermal Cycler (Bio-Rad, Hercules, CA, USA).
The lung sections were incubated with 5% bovine serum albumin (BSA) + 5% goat serum in phosphate-buffered saline (PBS) with 0.05% Tween-20 for 1 h at room temperature. The slides were then incubated with an anti-HSPG antibody (Invitrogen) at 4°C overnight. After washing, the slides were incubated with Fluorescein isothiocyanate (FITC) conjugated anti-rat antibody (Invitrogen) for 1 h at 37°C. Slides were counterstained with 4’,6-diamidino-2-phenylindole (DAPI).
Data were presented as the mean ± standard error. A student’s test was performed to confirm the significance of values, and P < 0.05 was considered significant.
This work was supported by the National Research Foundation of Korea [grant number 2017R1A5A2015061].
S. D. and M.-K. H are inventors of a patent related to this study. The authors have no conflicting interests.