E coli o111 b4

The neutralization of LPS by the peptides was assessed using a quantitative Chromogenic End-point Tachypleus Amebocyte Lysate (CE TAL) assay via the QCL-1000 kit (Xiamen, China). A constant concentration of LPS (1.0 EU/mL final concentration; E. coli, O111:B4, Sigma-Aldrich, USA) was incubated with various concentrations of the peptides or polymyxin B (PMB) (0–64 μg/mL final concentration; Sigma-Aldrich, USA) at 37°C for 15 min in the wells of pyrogenic sterile microliters plates. The 100 μL aliquots concentrate of limulus amebocyte lysate reagent was added and incubated at 37°C for 6 min. On the addition of chromogenic substrate, yellow color appeared. The reactions were stopped by adding 25% HCl, and then the absorbances measured at 540 nm (45 (link)).
The level of LPS in the plasma were detected using QCL-1000 kit (Xiamen, China) according to the manufacturer's instructions.

In vivo sepsis model was constructed using cecal ligation and puncture (CLP) method reported previously (Zhao et al., 2016 (link)). Firstly, the mice were anesthetized by isoflurane, local analgesine by 1% bupivacaine, and an incision was made in the midline. Secondly, the cecum was eviscerated, ligated using 5–0 silk suture, and punctured through-and-through using a 20-gauge needle. Thirdly, the abdomen was closed layer by layer. Finally, the mice in sham groups were manipulated in the same way, without the CLP component. All animal experiments were performed following the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
For in vitro analysis, the Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC, Manassas, United States). They were cultured in dulbecco’s modified eagle medium (DMEM) (HyClone, Logan, Utah, United States) containing 10% fetal bovine serum (FBS) (Biological Industries, Israel) and 1% penicillin-streptomycin (HyClone, United States), and then cultured at 37°C, 5% CO₂. The HUVECs were treated with lipopolysaccharide (LPS) (E. coli O111:B4, Sigma, St Louis, MO), HFn, GF9-HFn, HFn/Str, and GF9-HFn/Str.

Neutrophils were isolated from peripheral blood by centrifugation through a Ficoll-Hypaque gradient, sedimentation through dextran (3% wt/vol), and hypotonic lysis of erythrocytes. Neutrophils (5 × 10⁶ cells/ml, purity > 95%, viability > 98%, apoptotic < 2%) were cultured in RPMI 1640 medium supplemented with 10% autologous serum with human recombinant IFN-β (25–150 ng/ml, PeproTech) and then challenged with CpG DNA (1.6 μg/ml, E. coli strain B, Sigma), LPS (10 μg/ml, E. coli O111:B4, Sigma), serum amyloid A (10 μg/ml, PeproTech) with or without fludarabine (25 μM) or WP1066 (5 μM). In some experiments, neutrophils were first challenged with CpG DNA and treated with IFN-β at 1–4 h later. To study phagocytosis-induced apoptosis, neutrophils were cultured with live E. coli (American Type Culture Collection, ATCC 25922) at a ratio of 1:7 with or without IFN-β (50 ng/ml). At the designated time points, cells were processed as described below.

Mice were sedated with isoflurane, held upright and intranasally challenged with LPS (E. coli O111:B4; Sigma) at a concentration of 2.5 μg per gram of mouse in 0.9% saline (100 μl). The LPS challenge was given through the left nare to decrease liquid trapping in the nasopharyngeal dead space. Mice were maintained in the upright position until respirations returned to normal. Mice were monitored overnight prior to harvest 24 hr after challenge.