Lps e coli o55 b5

For BMDM generation, female C57Bl/6J mice, 7-10 weeks of age, were used
unless otherwise noted. For tolerance and septic shock experiments, male
C57Bl/6J mice, 6-10 weeks of age, were used. LPS (E. coli O55:B5; Sigma L2880)
and D-(+)-Galactosamine hydrochloride (Sigma G0500) were re-suspended in
sterile PBS and filter sterilized prior to intraperitoneal injection. For
in vivo infection experiments, mice were given
intraperitoneal injections of 1×10^7 CFU/kg of a GFP-expressing
Salmonella enterica serovar Typhimurium strain (SL1344)
suspended in PBS. Mice were maintained under specific pathogen-free conditions
in animal facilities at Columbia University Medical Center. All animal
experiments were carried out with the approval of the Columbia University
Institutional Animal Care and Use Committee, and in compliance with regulations
and guidelines set forth by Columbia University.

Tumor-free tissue from six patients who underwent lung tumor resection was used for the preparation of precision cut lung slices (PCLS) as described in detail previously³⁶ . Tissue was provided by the Asklepios Biobank for Lung Diseases (Gauting, Germany (project number 333–10)). All participants provided informed written consent, and the use of human tissue approved by the ethics committee of the Ludwig-Maximillian University (Munich, Germany (project number 455–12)). PCLS were cultivated in DMEM/Ham’s F12 medium supplemented with 0.1% FBS (Gibco, Life Technologies), 100U/ml penicillin-streptomycin and 2.5μg/ml amphotericin B (Sigma) at 37°C in 5% CO₂ atmosphere, and stimulated for 24h with 10μg/ml LPS (E. coli O55:B5, Sigma-Aldrich), 1ug/ml human LTβR-Ig fusion protein (kindly supplied by Jeffrey Browing, Biogen Idec), 20ng/ml recombinant human TNF (Cat. No. 300–01A, PeproTech) or 2 μg/ml agonistic antibody to human LTβR (clone BS1, Biogen Idec). Total RNA was isolated using peqGOLD Total RNA Kit (Peqlab).

Endotoxemia and CLP were performed according to Animal Resource Protocol approved by the Committee at the University of Rochester. For severe endotoxemia assay, 8–12-week-old C57BL/6 male mice were weighed and a single dose of 33 mg/kg LPS (E. coli O55: B5, Sigma-Aldrich) was administered by intraperitoneal (IP) injection to achieve LD70 mortality. For mild endotoxemia 8–12-week-old C57BL/6 male mice were weighed and 11 mg/kg of LPS was administered by intraperitoneal (IP) injection to achieve LD30 mortality. For CLP, caecum was ligated and punctured through with a 21-gauge needle. Mice were resuscitated with 1ml Ringers lactate injected subcutaneously. Animals were subsequently weighed daily until sacrifice to monitor disease progression and recovery.