Muramyl dipeptide mdp

Glucan was isolated from the yeast phase of C. albicans SC5314 using the method previously described by our laboratory (29 (link), 30 (link)). β-1,3-(D)-Glucan was produced in the laboratory of David L. Williams (TN, USA). Briefly, lyophilized C. albicans was extracted with 0.75 N NaOH (3x) and 0.5 N H3PO4 (2x) at 100°C for 15 min, followed by extraction with absolute ethanol (90°C) to remove lipids. The resulting product was a water insoluble particulate (~3µm) that was harvested and washed by centrifugation. The glucan was washed (3x) in dH2O, depyrogenated, sterilized, suspended in dextrose 5% (w/v) and water and stored at 4°C until used. The identity and structure of the glucan was confirmed by NMR (30 (link)). The product was >95% (1→3,1→6)- β-D-glucan. Sterility and endotoxin testing of the glucan was performed as described: there was no microbial contamination and no detectable endotoxin (30 (link)). Aliquots of (1→3,1→6)-β-D-glucan were prepared in sterile conditions and stored at 4°C.
Muramyl dipeptide (MDP) was obtained from InvivoGen Europe (Toulouse, France). MDP was dissolved in sterile water, aliquoted under sterile conditions, and stored at a temperature of -20°C.

Cells were treated with freshly prepared 25HC (50 µM, 8 h) (Steraloids, Rhode Island, USA) and the Nod2 activator muramyl dipeptide (MDP) (25 µg/ml, 8 h) (InvivoGen, California, USA). In some experiments, cells were pre-treated with either FAK inhibitor (5 µM) (PF-431396; Sigma Aldrich) or NFκB inhibitor (10 µM) (Bay-11–7082; InvivoGen) for 1 h or 30 min, respectively. Subsequently, these cells were either treated with various agents (25HC and MDP) or infected with viruses (RSV and IAV). Cells were infected with purified IAV or RSV at the multiplicity of infection (MOI) of 1. Virus adsorption was performed for 1.5 h (at 37 °C) in serum-free, antibiotic-free OPTI-MEM medium (Gibco). Following adsorption, cells were washed twice with PBS and infection was continued for an additional 8 h or 16 h in the presence of serum containing complete medium with or without treatment. For in vitro experiments (i.e., experiments with cultured cells), standard biological replicates of n = 4 were used.

Frozen PBMCs isolated from peripheral venous blood were thawed and differentiated to PBMDMs, as described earlier. Transduced PBMDMs were cultured in 24-well plates (OptiPlate-24, White Opaque 24-well Microplate) in 0.4 mL medium containing 1 mM luciferin. Cells were stimulated with 200 ng/mL LPS and luminescence detected over time in a CO₂ Lumistar Omega luminometer. Post-assay, pro-viral copies were measured by qPCR, using a Lenti-X™ Provirus Quantitation Kit (Clontech; Oxford, UK). For in vitro stimulation, other ligands used included human recombinant Interleukin-1β [IL-1β](PeproTech), Flagellin FliC from Salmonella typhimurium (NovusBio; Littleton CO, USA), muramyl-dipeptide [MDP] (InvivoGen; Toulouse, France), and LPS extracted using modified phenol/water method (27 (link)) from IBD mucosa-associated E. coli isolates, LF82 and LF10 (28 (link), 29 (link)).

Assays were carried out with approximately 1 to 2 × 10⁴ cells/well of microglia seeded in 24-well tissue culture plates. At the time of the assay, the culture medium was removed and replaced with fresh medium without antibiotics, and the cells were pretreated with the pertinent inhibitors for 2 h prior to adding B. burgdorferi (MOI of 10:1). The microglial cells were incubated with the bacteria and inhibitors for a further 24 h, followed by collection of supernatant after centrifuging at 2,095 × g for 10 min at 4°C. Supernatants were stored at −20°C until analysis. The following inhibitors were used: SB203580 and BIRB796 (p38); U0126 (MEK1/2); SP600125 and Jun N-terminal kinase (JNK) inhibitor VIII (JNK) (all but one were from EMD Millipore, Billerica, MA; BIRB796 was obtained from Cayman Chemical Co., Ann Arbor, MI); and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) (TLR2/4), CLI-095 (TLR4), Gefitinib (RipK2), and myeloid differentiation primary response 88 (MyD88) inhibitory peptide (InvivoGen, San Diego, CA).
Pam3CSK4 (Imgenex, San Diego, CA), LPS O55:B5 (Sigma-Aldrich, St. Louis, MO), or muramyl dipeptide (MDP, InvivoGen) were included as positive control agonists when required.

Caco2 cells (ATCC) were cultured in Dulbecco's modified Eagle medium (HyClone) supplemented with 20% fetal calf serum (HyClone), 2 mM L-glutamine, 100 U/ml Penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO2. The cells were used between passages 15 and 30. For all of the experiments, to better mimic the steric conditions existing in the intestine in vivo, the cells were plated at a subconfluent cell density onto 6-well Millicell hanging filter inserts (3 μm pore size, Polyethylene Terephthalate, Millipore) that allow free access of media to their apical and basolateral sides. Media were changed every 24 h. To determine the role of NOD2, fibroblast growth factor 9 (FGF9) (10 ng/ml; R&D Systems), a high affinity ligand for the fibroblast growth factor receptor-3 (FGFR-3); muramyl dipeptide (MDP) (10 μg/ml; InvivoGen), an agonist for intracellular NOD2; and FGF9 (10 ng/ml) plus MDP (10 μg/ml) were added to both sides of the inserts daily starting at 24 h post-plating and ending at 72 h post-plating.

Ten dissected nerve cords were collected per condition. Connectives between ganglia were injured in a standard manner using a pair of sterilized fine iridectomy scissors. Axotomized nerve cords were separately incubated in L-15 media containing different microbial components: 3 × 107 CFU/ml of heat-killed Gram positive (M. nishinomiyaensis) or negative (A. hydrophila) bacteria, 100 ng/ml of E. coli LPS (0111:B4 strain, Invivogen), 100 μg/ml of zymosan (Invivogen), 10 μg/ml of Muramyl dipeptide (MDP, Invivogen), 2 μg/ml of lipoteichoic acid (LTA) (Invivogen) or 10 μg/ml of Poly(I:C) (Invivogen) for different time (T0, t = 6 h) at room temperature. The supernatant of culture of cells infected with Vesicular stomatitis virus (VSV) for several days was used to stimulate the axotomized nerve cord ex vivo. Incubations without microbial components were performed in the same conditions as controls (H₂O).