Lps from escherichia coli o127 b8

Human monocytic leukemia (THP-1) cells (ECACC collection; Sigma-Aldrich, St Louis, MO, USA) were maintained in culture in Roswell Park Memorial Institute medium (RPMI 1640, Lonza, Basel, Switzerland) supplemented with 1% L-glutamine, 10% heat-inactivated FBS and 1% penicillin–streptomycin. Cells were kept viable at 37 °C and 5% CO₂.
To differentiate into macrophages, THP-1 cells (1x10⁶ cells/mL) were seeded in 24 well plates and treated with 100 nM phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich, St Louis, MO, USA) for 72 h at 37 °C, 5% CO2. After incubation, non-attached cells were aspirated, and adherent cells were treated with peptides (0.5 µg/mL), peptide network (0.5 and 5 µg/mL) or remained untreated. Docosahexaenoic acid (DHA) at 32.8 µg/mL was used as a positive control [39 (link)]. Following incubation for 24 h, LPS from Escherichia coli O127:B8 (Sigma-Aldrich, St Louis, MO, USA) was added at 100 ng/mL for 24 h at 37 °C, 5% CO₂. Cell supernatants were collected and TNF-α concentrations were determined by ELISA according to the manufacturer’s instructions (BioLegend, San Diego, CA, USA). Absorbance was measured at 450 nm with a microplate spectrophotometer (SpectraMax M3, Molecular Devices, Sunnyvale, CA, USA).

Dimethylsulfoxide (DMSO) and LPS from Escherichia coli O127:B8 were obtained from Sigma-Aldrich (St. Louis, MO, USA). LPS is the major structural component of the outer wall of all Gram-negative bacteria and recognized by TLR4. TCDD (>99% purity) was originally obtained from Dow Chemical Co. (Midland, MI, USA). Other molecular biological reagents were purchased from Qiagen (Valencia, CA, USA) and Roche Clinical Laboratories (Indianapolis, IN, USA).

Immediately after radon inhalation, LPS (LPS from Escherichia coli O127:B8 Sigma-Aldrich, Tokyo, Japan) at a dose of 1.0 mg/kg body weight was intraperitoneally administered once to the mice in the Rn + LPS group. Mice in the LPS group received the same dose, while those in the sham group did not receive LPS.

LPS from Escherichia coli, (O127:B8) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Rabbit anti–ACE, anti–ACE2, and anti–p65, and mouse anti–phospho–p65 and anti–IκBα antibodies were procured from Santa Cruz Biotechnology (Delaware, CA, USA). Rabbit anti–p38 MAPK, anti–phospho–p38 MAPK, anti–ERK1/2, anti–phospho–ERK1/2, anti–stress–activated protein kinase (SAPK)/JNK, anti–phospho–SAPK/JNK, horseradish peroxidase (HRP)–conjugated secondary antibody goat anti–rabbit IgG, and horse anti–mouse IgG antibodies were all purchased from Cell Signaling Technology (Danvers, MA, USA). TNF–α and IL–1β kits were purchased from Invitrogen (Eugene, OR, USA). AngII and Ang–(1–7) enzyme–linked immunosorbent assay (ELISA) kits came from Kamiya Biomedical (Seattle, WA, USA). Mas receptor blocker A779 was obtained from AbBiotech (San Diego, CA, USA). SB203580 (a specific inhibitor of p38 MAPK), PD98059 (a specific inhibitor of ERK1/2) and SP600125 (a specific inhibitor of JNK) were all purchased from Santa Cruz Biotechnology (Delaware, CA, USA).

All animal procedures complied with the National Institute of Health guidelines and were reviewed and approved by the local Hospital Animal Care and Use Committee (IACUC approval number 2016082901). Ten-week-old male C57BL/6 mice were purchased from BioLasco Biotechnology (Taipei, Taiwan). Animals were initially anesthetized through an intraperitoneal injection of a 0.01 mL/kg mixture (1:1 volume/volume) of tiletamine-zolazepam (Zoletil^®; Virbac, Carros, France) and xylazine hydrochloride (Rompun^®; Bayer HealthCare AG, Leverkusen, Germany), and subsequently subjected to intrafemoral injections of 10 mg/kg LPS (from Escherichia coli O127:B8; Sigma-Aldrich, St. Louis, MO, USA) or 20 mg/kg LTA (from Staphylococcus aureus; Sigma-Aldrich) in phosphate-buffered saline (PBS; 10 μL). Seven to ten days post-injection, mice were sacrificed (5–7 mice per group). The inoculated femur underwent immediate 10% formaldehyde fixation and was subjected to micro-CT analysis.

C57BL/6 and C57BL/6 Caspase-1/11-deficient mice were purchased from Institut Pasteur de Montevideo. BMDCs were generated by differentiation of bone marrow precursors from 8- to 10-week-old C57BL/6 and KO mice for 10 days in the presence of 20 ng/mL recombinant mouse GM-CSF (PeproTech) as described elsewhere^{54 (link)}. The cells obtained were between 85 and 95% CD11c⁺. On day 10, cells were plated at 0.4 million per well in 96-well plates and stimuli were added in medium containing 5 ng/mL GM-CSF. BMDC were primed with 10 ng/mL lipopolysaccharide (LPS from Escherichia coli O127:B8, Sigma) for 2 hours and then stimulated with different doses (10, 2.5 and 0.5 µg/mL saponins concentration) of QB-90 or IMXQB-90. Alum (Alhydrogel, Sigma) was used as a control for inflammasome activation. After 3 hours, cell response was measured in terms of IL-1β production. Cell culture supernatants were assayed for IL-1β with ELISA kits from Biolegends according to the manufacturer’s instructions. Cell viability was evaluated at the end of the assay by flow cytometry, using Topro-3 staining (Invitrogen).

Wistin was purchased from Indofine Chemical Company, Inc. (Hillsborough, NJ, USA) and was dissolved in dimethyl sulfoxide (DMSO). Dulbecco’s modified Eagle’s medium (DMEM, high glucose) was purchased from Hyclone Laboratories Inc. (Marlborough, MA, USA), and fetal bovine serum (FBS) was purchased from Corning (Corning, NY, USA). Penicillin-streptomycin-glutamine was purchased from Gibco (Waltham, MA, USA). MTT, LPS from Escherichia coli O127:B8, N-(1-naphtyhyl) ethylenediamine dihydrochloride, phosphoric acid, sulfanilic acid, and nitrite ion standard solutions were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco’s phosphate-buffered saline (DPBS) and Hank’s balanced salt solution (HBSS) were purchased from WELGENE, Inc. (Gyeongsan, Korea). 2′7-Dicholrodihydrofluorecsein diacetate (DCF-DA) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The quantitative reverse transcription polymerase chain reaction (qRT-PCR) primers were purchased from Macrogen (Seoul, Korea). Primary antibodies ((iNOS (#13120), COX-2(#12282), phospho-p65 (#13346), phospho-JNK (#9255), phospho-ERK (#4370), and β-actin (#8457)) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Goat anti-rabbit IgG (5220-0036) and goat anti-mouse IgG (5220-0341) antibodies were purchased from SeraCare Life Sciences, Inc. (Gaithersburg, MD, USA).