Tak 242

LPS (Eb-ultrapure, 0111:B4), Pam3CSK4, pepinhTRIF, and TAK-242 were obtained from InvivoGen, nigericin was obtained from Invitrogen, and Bacillus anthracis PA was obtained from List Biological Laboratories. Colchicine was obtained from Sigma, and VX-765 was obtained from Sellekchem. BAA-473 was a gift from Dr. Canham (Novartis). DRAQ5 was purchased from eBioscience. TNFα, IFN-β, IFN-γ, M-CSF, IL-3, IL-4, and IL-10 were purchased from Immunotools. TcdA and TcdB from C. difficile strain VPI10463 were recombinantly produced and supplied by Prof. Ralf Gerhard [40 (link)]. Both toxins are identical to TcdA and TcdB from strain cdi630, which was used for infection assay. The HTRF kits for human IL-1β and TNFα were obtained from Cisbio; the ELISA kit for human and mouse IL-1β was obtained from R&D Systems. Both were used according to the manufacturer’s instructions. For the C. difficile supernatant transfer, the following reagents were used: Butyric acid (Sigma-Aldrich: W222119-1KG-K), Various amino acids (Roth or Sigma), Iron sulfate heptahydrate (Sigma-Aldrich: 215422-250G), Triton-X 100 (Roth: 3051.2), M-Per (Sigma-Aldrich: 78501), Protease inhibitor (Sigma-Aldrich: 11836170001).

Primary human ONH LC cells were grown to confluency and pretreated with the selective TLR4 inhibitor, TAK-242 (In vivoGen, San Diego, CA, USA) at 15mM for 2 hours. TAK-242 is a cyclohexene derivative that specifically inhibits TLR4 signaling by binding to the intracellular domain of TLR4 and blocking downstream signaling. We previously performed a dose response curve using TAK-242 in primary human trabecular meshwork cells in culture and determined 15μM had the greatest inhibitory effect without affecting cell viability (29 (link)). Cells were then treated with TGFβ2 (5ng/mL) and/or TAK-242 (15μM) for 72 hours in serum-free medium. For TLR4 activation studies, hONH LC cultures were grown to confluency and treated with cellular fibronectin (cFN) (10μg/mL) containing the FN-EDA isoform (F2518; Sigma-Aldrich Corp., St. Louis, MO, USA) and/or TGFβ2 (5ng/mL), and/or TAK-242 (15μM) for 72 hours in serum-free medium. Western blot and immunocytochemistry experiments were performed as described below.

The TLR4 agonist LPS (055 : B5, Sigma-Aldrich) was dissolved in sterile saline and was administered at 1 μg/10 μl [32 (link)] into the lateral ventricle. The TLR4 antagonist TAK-242 (CLI-095, InvivoGen) was dissolved in 10% dimethyl sulfoxide (DMSO, D2650, Sigma-Aldrich) and was infused via intracerebroventricular injection at a dose of 0.6 mg/10 μl [33 (link), 34 (link)]. Drugs were infused by a Hamilton syringe (80630, Hamilton Co., Reno, USA) with a rate of 1 μl/min, and the needle was left for an additional 10 min before withdrawal to prevent reflux. All the infusion procedures were conducted 30 min before treatment every day.

Anti-GAPDH and anti-rabbit were purchased from Sigma-Aldrich Co. (St. Louis, MO, United States). DMEM/F12 culture medium, fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco BRL (Carlsbad, United States). Anti-ICAM and anti-VCAM were purchased from Santa Cruz Biotechnology (Dallas, United States). Anti-RP-1 was obtained from BD Biosciences (San Jose, CA, United States). Histopaque-1083 and histopaque-1119 were purchased from Sigma-Aldrich Co. (St. Louis, MO, United States). Organic and inorganic compounds, salts, acids, and solvents were purchased from Merck (Darmstadt, Germany). LPS (Ultra pure lipopolysscharide from E Coli 0111:B4 strain-TLR4 ligand) Cat. code TLRL pelps, was purchased from Invivogen (San Diego, Ca, United States), TAK-242, and Collagenase type II were purchased from Invivogen (San Diego, United States). Sterile plastic material for obtaining and culturing cells was purchased from Falcon (Ballerica, Ca, United States). Xylazine 2% was purchased from Laboratorios Centrovet (Santiago, Chile). Ketamine 100 mg/ml was purchased from Richmond Vet Pharma (Bs. As., Argentina). Interferon-beta (IFN-β) was purchased from InterferonSourse (NJ, United States). MACS^® culture medium for neutrophil isolation was purchased to Miltenyi Biotec (Teterow, Germany).