Lipopolysaccharides (lps)

The porcine small intestine epithelial cell line SIEC02 (Wang et al., 2014 (link)) was kindly provided by Prof. YM Zhang (Northwest A&F University, Yangling, PR China) and cultured in complete culture solution (DMEM:F12 medium = 1:1, 10% FCS; Gibco BRL, Life Technologies, Grand Island, NY, USA). When the cells reached 80–90% confluent, they were exposed to LPS (Sigma-Aldrich) at the indicated concentrations. The negative control was incubated with DMEM:F12 medium and three parallel replicates were used for each group. To determine the role of TWEAK-independent and TWEAK-dependent Fn14 signaling in LPS-induced intestinal inflammation, 10⁶ porcine small intestinal epithelial cells were seeded into each well of a 24-well plate and stimulated with LPS for 24 h. Then, recombinant TWEAK (Abcam, Cambridge, MA, USA), Fn14 antibody (Abcam), or a TNF-α antibody (Abcam) was added at the indicated final concentrations and the cells were collected for analyses after 24 h incubation. Isotype control IgG was used as control in multiple blocking antibodies experiments. Small interfering RNA (siRNA)-mediated knockdown of Fn14 gene in SIEC02 was also used to determine the role of Fn14 in the present culture system.

ADSCs at passage 3 were grown in 6-well plates (3 × 10⁴/well). After overnight culture, the diabetic microenvironment pretreatment group was co-cultured in two different concentrations of stimuli (low concentration precondition: LPS (Abcam, MA) 1 µg/mL, AGE (Abcam) 1 µg/mL, and glucose (Proteintech, Wuhan, China) 2.5 mg/ml; high concentration precondition: LPS 2 µg/mL, AGE 5 µg/mL, and glucose 4.5 mg/mL). After incubating for 24 h, 48 h, and 72 h, the medium was changed to low-sugar DMEM containing 10% FBS without stimulation, as in the normal control group.

hiPSC-MGs were stimulated with LPS (100 ng/ml; Sigma-Aldrich) for 8 hours and followed by a washout. Cell lysates were collected at 4, 8, and 12 hours after washout, respectively, to examine the dynamics of NLRP3 and p62 by Western blotting. For testing the dynamics of NF-κB signaling by immunochemistry, cells after LPS washout were fixed at 8 and 12 hours, respectively, and stained with anti–NF-κB (Abcam) and anti-LC3B (Enzo Lifesciences) and observed in a Zeiss LSM confocal microscope. The details of the dilution for individual antibodies are mentioned in table S4.
For experiment with secondary stimulus, both LPS-primed and untreated mC9-MG and isoC9-MG were stimulated with 1 mM ATP for 30 min, and NLRP3 intensity was first measured at 30 min and also following a washout interval of 12 hours.
For assessing NF-κB activation, the Carl Zeiss image files were imported into Imaris version 8.0.2; the nuclear region was identified using DAPI staining and the intensity of the nuclear NF-κB was measured in voxels using Imaris software. Results were exported from Imaris in CSV formats, and graphs represent the colocalized voxels of DAPI and nuclear NF-κB.

The ability of Cur-SHAP to suppress LPS-induced ROS generation was measured using a DCFDA-cellular ROS assay kit (ab113851, Abcam). Briefly, C2C12 myoblasts were cultured in 96-well plates at a density of 10⁴ cells/well and incubated for 24 h. After the medium was supplemented with 1 mg/mL of Cur-SHAP for 24 h, 1 μg/mL LPS (L2880, Sigma) was added to induce ROS generation for 24 h. The medium was then removed, washed with PBS 2 times, and cultured in medium with 25 μM DCFDA reagent for 45 min at 37 °C. The fluorescence signal was detected by multimode microplate readers (Molecular Devices, SpectraMax i3x, USA) with emission and excitation wavelengths of 535 nm and 485 nm, respectively.

Immunohistochemical staining and quantitative image analysis were performed as previously described [39 (link)]. Briefly, slides were incubated with antibody to CXCL10 (Rabbit; 4:100, OriGene, Rockville, Maryland), LPS (Mouse; 1:100, Abcam, Cambridge UK) or myeloperoxidase (MPO) (Rabbit; 1:1000, DAKO (Agilent) Santa Clara, CA) then rabbit or mouse Polink- 2 horseradish peroxidase (HRP) and developed with Immpact DAB (3,3′-diaminobenzidine; Vector Laboratories, Burlingame, CA), or red-AP (Vector Laboratories) then counterstained and mounted in Permount (Thermo Fisher, Waltham, MA), and scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies, Vista, CA), yielding high-resolution data from the entire tissue section. Representative regions of interest (500 mm²) were identified and high-resolution images extracted from these whole-tissue scans. The percentage area positive for myeloperoxidase (MPO) (a neutrophil marker), LPS and CXCL10 was quantified using Photoshop CS5 and Fovea tools.
Liver biopsy samples from people without HIV, HBV and HCV infection who were undergoing liver biopsy for other indications, were used as controls for CXCL10 immunohistochemical staining.

Mice transgenic for the human ACE2 receptor (K18-hACE2, B6.Cg-Tg(K18-ACE2)2Prlmn/J, 034860, The Jackson Laboratory) were exposed to 20 μg LPS (MilliporeSigma) or LPS 20 μg and 10 μg SARS-CoV-2 S1 spike protein (Abcam) i.n. to establish the model of spike protein–induced pulmonary injury. hACE2-negative litter mates served as controls. In a separate series of experiments, hACE2-positive mice were treated with both LPS and spike protein applied i.n., with one group of animals receiving reparixin (15 μg/g BW i.p.) 1 hour before and 2 hours after LPS/spike protein challenge (50 (link), 68 (link)). Clinical scoring was performed by a scientist blinded to treatment. Mice were sacrificed after 24 hours, and lungs were removed and either processed for histopathological analysis (see below).