Il 1 receptor antagonist

Human Caco-2 and T84 intestinal epithelial cells as well as the monocytic THP-1 cell line were obtained from the American Type Culture Collection (Manassas, VA) and cultured according to standard protocols in 95% air with 5% CO₂ at 37°C [45 ]. Lentiviral transduction of Sigma MISSION® shRNA against HMOX1 (TRCN0000290436) and a control non-targeting shRNA (SHC216) was performed using established protocols (University of Colorado Functional Genomics Facility, Aurora, CO) [46 (link)]. THP-1 cells were exposed to bacteria at an MOI of 10:1. IL-1 receptor antagonist (Anakinra, Amgen, Thousand Oaks, CA) was used at a concentration of 100 ng/mL. Polarized IECs were grown on Transwell inserts with 0.4 μm pore size (Corning, Tewksbury, MA) and cultured at least overnight with THP-1 cells for co-culture experiments.

All drugs were injected intrathecally in 10ul of saline under isoflurane analgesia. The following drugs were used; LY303870 (20ug), a SP neurokinin 1 (NK1) receptor antagonist (Eli Lily Company); CGRP_8-37 (20ug), a CGRP receptor antagonist (Sigma); etanercept (100μg), a TNF inhibitor (Pfizer); anakinra (100ug), an IL-1 receptor antagonist (Amgen); TB-2081 (20ug), an IL-6 receptor antagonist (a generous gift from Dr. Kenner Rice, National Institute on Drug Abuse); RS504393 (3ug), a C-C chemokine receptor type 2 (CCR2) antagonist (Sigma); muMab 911 (12.4ug), an anti-NGF antibody (Pfizer); and PD98059 (20ug), a selective inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) phosphorylation (Sigma).