L2630

Venous blood was collected from human volunteers by venipuncture into EDTA vacutainers. PBMCs were isolated by density gradient centrifugation using Lympholyte-poly Cell Separation Media (Cedarlane, CL5071). Monocytes were then purified from isolated PBMCs by negative selection using a Human Monocyte Isolation Kit (STEMCELL Technologies, 19359) and cultured for 7 days in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, 11875119) with 10% FBS, antibiotics, and human recombinant GM-CSF (20 ng/mL) (R&D Systems, Bio-Techne, 215-GM-010). Cells then were washed with PBS to remove nonadherent cells and detached by incubation in TrypLE Select at 37°C followed by gentle cell scraping. Macrophages were plated in 96-well plates at a density of 5 × 10⁴ cell/well and allowed to adhere overnight. Human macrophages were then polarized to an M1 activation state with 30 ng/mL human recombinant IFN-γ (R&D Systems, Bio-Techne, 285-IF-100) 100 ng/mL LPS (MilliporeSigma, L2630) for 24 hours before use.

Mouse macrophage cell line RAW 264.7 was cultured in DMEM medium (cat. # 21885-025, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat inactivated fetal bovine serum (cat. # 10270-106, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (cat. # 15070-063, Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were cultured in 37 °C and 5% CO₂. Each compound was diluted in Carbowax^TM 400 + 10% ethanol (cat. # 1.00983, Merck, Darmstadt, Germany), used also as control solvent. Final concentration in culture was 0.1% v/v carbowax and 0.01% v/v ethanol. RAW 264.7 macrophages were activated using 100 ng/mL lipopolysaccharide (LPS) (L2630, Merck, Darmstadt, Germany). In IC₅₀ determination experiments, macrophages were pre-treated for 1 h with the respective compound prior to LPS stimulation.

Mouse bone marrow macrophages (BMMs) were prepared and cultured according to previously described methods [20 (link)]. Briefly, isolate the femur and tibia of the mouse after anesthesia, cut off both ends of the bone, and rinse with cell culture, and then, centrifuge (500 × g, 10 minutes, 4°C) to collect bone marrow cells (BMs). Next, we used 10 ng/mL recombinant mouse macrophage colony-stimulating factor (cyt-439, AmyJet Scientific, China) to induce BMs to differentiate into macrophages (BMMs). In the present study, BMMs experienced stimulation with different stimuli: (1) BMMs were treated with curcumin (10 μmol/L) for 1 hour and then incubated with 100 mg/mL LPS (L2630, Merck, USA)+20 ng/mL IFNγ (I4777, Merck, USA) for 24 hours, and (2) BMMs were treated with 10 μmol/L Compound C (CC, HY-13418A, MCE, USA) for 2 hours and then incubated with curcumin (10 μmol/L) for 1 hour, followed by 100 mg/mL LPS+20 ng/mL IFNγ challenge for 24 hours.

RAW264.7 murine macrophage cells were obtained from the National Centre For Cell Science (NCCS), Pune, India. The cell lines were cultivated and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, AT151; HiMedia, India) supplemented with 10% fetal bovine serum and 100 μg/mL of penicillin–streptomycin (15,140,122; Invitrogen, USA) at 37 ºC in a 5% CO₂ incubator. For immunostimulation studies, RAW264.7 cells were exposed to different concentrations of Y-SFF and O-SFF for 48 h followed by an assessment of various cellular and biochemical markers. This relatively longer duration of stimulation was chosen to understand the chronic effects of SFF exposure that could also be implicated in its overall safety. For immunomodulatory studies, RAW264.7 cells were first exposed to Y-SFF and O-SFF at respective concentrations for 48 h followed by stimulation with 1 μg/mL of lipopolysaccharide (LPS) (Merck; L2630) for 24 h, and subsequent assessment of various cellular and biochemical parameters. Control cells (C) and LPS-only treated cells were also maintained in parallel.

To induce endotoxemia, a model that mirrors the lung injury observed in polymicrobial sepsis, mice were challenged with sublethal LPS (#L2630, Sigma-Aldrich, St. Louis, MO, USA) intraperitoneally at 10 mg/kg and sacrificed at 24 and 72 h for blood and tissue harvesting [15 (link)]. To degrade NETs, mice were treated with an intraperitoneal injection of 100 μL of DNase I (1 mg/mL dissolved in saline, 143582, Roche, Basel, Switzerland) starting 1 h after LPS administration and the process was repeated daily until the termination of the experiments [16 (link),17 (link),18 (link)]. Mice were randomly assigned to treatment groups with approximately equivalent numbers of mice in each group.

To evaluate the inflammatory response and induce endotoxemia, the mice were injected with lipopolysaccharides (LPS—Escherichia coli serotype O111:B4. L2630, Sigma-Aldrich, St. Louis MO, USA) or saline solution (0.9%).
LPS was diluted in saline solution, and the concentration was adjusted according to the protocol (5 or 12 mg/kg). It was administered intraperitoneally, and after 2 h, the mice were sacrificed to collect the tissues (hypothalamus, spleen, liver, and bone marrow cells).

Mouse models were induced using modified protocols as previously reported [7 (link)–9 ]. Mice were randomly grouped and sensitized on day 1 and day 8. For the OVA/Alum group of asthma, 25 µg OVA (A5503, Sigma Aldrich, St. Louis, MO, USA) dissolved in 100 µl 0.9% saline was mixed 1:1 with Imject Alum (Pierce, Rockford, IL, USA) by intraperitoneal (IP) injection. For the OVA/CFA group of asthma, 25 µg OVA dissolved in 100 µl 0.9% saline was mixed 1:1 with CFA (F5881, Sigma Aldrich, St. Louis, MO, USA) by IP. injection. For the OVA/LPS group of asthma, mice were lightly anesthetized with isoflurane, and intratracheally injected using a combination of 25 µg OVA with 0.1 or 10 µg LPS (L2630, Sigma Aldrich, St. Louis, MO, USA) in a total volume of 40 µl, with 0.9% saline as the diluent. For the OVA/CFA/0.1 LPS group, the model of sensitization is a combination of the OVA/CFA group and OVA/LPS group. After sensitization, the above-mentioned groups were challenged with aerosolized 1% OVA for 30 min on days 15-17. For the OVA/CFA/0.1 LPS + DNase I group or the OVA/CFA/0.1 LPS + CI-amidine group, on the basis of the OVA/CFA/0.1 LPS group, intravenous injection of DNase I (5 mg per kg body weight) or intraperitoneal injection of Cl-amidine (10 mg per kg body weight) was performed 1 h before each challenge. Mice sensitized and challenged with 0.9% saline were used as controls.