Gts 21

GTS-21 was obtained from Abcam (Cambridge, MA, USA). The pH of the GTS-21 solution was adjusted to 7 before intra-peritoneal injection in mice. 0.9% normal saline solution was used as the control vehicle.

Lung organoids were established based on a previous report with modifications (48 (link)). Briefly, freshly sorted lineage-labeled cells (CD45^–CD31^–EPCAM⁺tdTomato⁺) were resuspended in basic media (advanced DMEM/F12 supplemented with 10% FBS and mixed with lung mesenchymal cells (CD45^–CD31^–EPCAM^–) at a ratio of 1:6, followed by resuspension in growth factor–reduced Matrigel (BD Biosciences) at a ratio of 1:5. A 10 μL mixture was placed in a prewarmed 24-well Transwell insert with a 0.4 mm pore (Corning) or 48-well cell culture plate at 37°C for 20 minutes. Approximately 2 × 10³ to 5 × 10³ Sftpc⁺ cells were seeded in each insert. 500 μL or 250 μL organoid media (listed in Supplemental Table 2) was placed in the lower chamber, and medium was changed every 3 days with or without LPS (1 μg/mL, Sigma-Aldrich), GTS-21 (10 μmol/L, Abcam), MLA (10 μmol/L, Abcam), WNT7B (100 ng/mL, referred to in refs. 49 (link), 50 (link); Novus Biologicals). R-Spondin-1 (500 ng/mL, Peprotech) and ROCK inhibitor Y-27632 (5 μmol/L, APExBio) were added in the medium for the first 3 days of culture. Colony-forming efficiency and size of organoids were analyzed at day 10 after plating if there is no specific description.

The in vitro organ culture was performed following a previously published report (Kreiss et al., 2004 (link)). Briefly, the stomachs were removed, opened, and washed three times with RPMI 1640 medium containing 100 μg/ml streptomycin and 100 U/ml penicillin under sterile conditions. The tissue was pinned on a Sylgard plate, and the muscularis was then further dissected, weighed and placed in individual wells of 24-well polystyrene plates in the absence or presence of α7nAChR agonists (PNU or GTS-21) or antagonist methyllycaconitine citrate (MLA). GTS-21 and MLA were purchased from Abcam (Cambridge, UK). After an incubation period of 2 h in RPMI 1640 at 37°C and 5% CO₂, the cultured tissue was inspected for contamination and cultured for 2 h. The cultured tissue was blotted dry, immediately quick-frozen in liquid nitrogen and stored at –80°C.

Cells were suspended in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in a 5% CO₂ atmosphere. The PBMCs (1×10⁶ cells/ml) were cultured for 72 h in 24-well plates and subsequently stimulated with anti-CD3 (3 μg/ml, clone HIT3a; BD Biosciences, Franklin Lakes, NJ, USA) and anti-CD28 (3 μg/ml, clone CD28.2; BD Biosciences) antibodies in the presence or absence of different concentrations (0.01, 0.1 or 1 μmol/l) of GTS-21 (Abcam, Cambridge, MA, USA). Purified CD4⁺ T cells (1×10⁶ cells/ml) were stimulated using anti-CD3-coated 96-well plates (BioCoat™ anti-human CD3 T-cell activation plates; BD Biosciences) plus anti-CD28 antibodies (3 μg/ml), in the presence of IL-12 (15 ng/ml, Peprotech, Inc., Rocky Hill, NJ, USA) and anti-IL-4 antibodies (4 μg/ml, Peprotech, Inc.) for Th1 differentiation for 72 h with GTS-21 (1 μmol/l) alone or combined with α-bungarotoxin (αBgt, 1 μmol/l; Sigma, St. Louis, MO, USA).

Male BALB/c mice (24–30 g) were purchased from CAVENS animal center (Chang Zhou, China, SCXK (SU) 2011-0003). The mice were housed in the specific pathogen-free (SPF) facility at Nanjing University. All protocols were approved by the Nanjing University of Science and Technology Animal Care and Use Committee. In addition, the animals received humane care in compliance with the Principles of Laboratory Animal Care.
BALB/c mice were randomly divided into four groups (8 mice/each group): Group I (Control, Ctrl): mice drunk sterile tap water freely and were intraperitoneally injected with saline daily from day 0 to day 7; Group II (DSS group): mice received 3.5% DSS (molecular weight 36–50 kDa, MP Biomedicals, USA) in drinking water freely from day 1 to day 7 and were intraperitoneally injected daily with saline from day 0 to day 7; Group III (GTS-21 group): mice were intraperitoneally injected daily with GST-21 (10 mg/kg/day, sigma, USA) from day 0 to day 7 and received 3.5% DSS in drinking water freely from day 1 to day 7; Group IV (α-BGT group): mice were pre-treated with α-BGT (0.1 mg/kg/day, abcam, USA) via intraperitoneal injection 30 min prior to GTS-21 injection from day 0 to day 7 and received 3.5% DSS in drinking water freely from day 1 to day 7. Animals were euthanized on day 8.

Groups of mice were treated with GTS-21 (provided by Abcam, 4 mg/kg) or vehicle (sterile saline) intraperitoneally 12h after CLP procedure and repeated every 12h respectively. At indicated time points, animals were sacrificed, and blood, peritoneal lavage fluid (PLF), and spleen, lung, and liver samples were collected for further evaluation. For survival analysis, mice were treated with GTS-21 or vehicle beginning 12h after CLP procedure and continued twice daily for three consecutive days. Survival was routinely evaluated for 7 days.

Mice were intranasally (i.n.) administered with recombinant mouse IL-33 (0.5 µg/dose, R&D, California, USA) in mice intraperitoneally (i.p.) administered with PNU-282987 (20 mg/kg, Abcam, California, USA) or GTS-21 (20 mg/kg, Abcam, California, USA) over three consecutive days (22 (link)). For Alternaria experiments, mice were i.n. administered with AA (100 µg/dose, Greer Labs, Lenoir, North Carolina, USA) in the presence or absence of PNU-282987 or GTS-21 on four consecutive days. Mice were sacrificed on the second day after the last challenge.