Ultrapure tlr4 specific lps

Three adult females from each genotype were housed with floxed fertile males from respective backgrounds overnight in separate cages. Successful mating (day 1 of pregnancy) was defined as the morning of finding the presence of a vaginal plug. Plug-positive females were housed separately until processed for experiments. Litter size, pregnancy rate and outcomes were monitored in timed pregnant mice. Ultrapure TLR4-specific LPS (2.5 μg/mouse and 40 μg/mouse, InvivoGen) was administered intraperitoneally (i.p.) on day 16 of pregnancy. Parturition was monitored from days 16 through 21 by daily observation of mice in the morning, noon and evening. Birth timing was defined by the observation of the first pup born. PTB was defined as birth occurring earlier than day 19 of pregnancy (day 1 = vaginal plug in the morning). Resorption sites and placental scars were identified in dams showing preterm or difficult deliveries by examination of the uterus. The number of pups/masses delivered was compared with the number of resorption sites and placental scars. Anti-IL10 antibody (500 μg/mouse, i.p., custom made, JES5–2A5) (Clemente-Casares et al., 2016 (link)) was injected in both p53^f/f and Pgr^Cre/+p53^f/f mice 30 min before and after LPS injection (1 mg/mouse, i.p.). At least three independent mice in each genotype were used for biochemical and immunostaining assays.

Mice were anesthetized with inhaled isoflurane and all reagents were administered via intratracheal aspiration (21 (link)). To measure innate immune responses, mice were administered a single dose of 100 μg of Dermatophagoides pteronyssinus (Der p) extracts (Greer Laboratories, Lenoir, NC) or 1 μg of TLR4-specific ultrapure LPS (Invivogen, San Diego, CA) in a total volume of 40 μl, and bronchoalveolar lavage (BAL) was performed at 2, 4, 6, 8 or 24 hours after challenge. For the model of HDM-mediated asthma, mice were given 100 μg Der p extracts on days 0, 7, 14, and BAL performed on day 17. For the OVA/LPS model of asthma, mice were given 100 μg LPS-depleted OVA (BioVendor Inc. Candler, NC) together with 100 ng LPS from E. coli (Sigma, St. Louis, MO) on days 0, 7, and 14 and BAL was performed on day 16. Cell differential analysis was carried out as described previously (21 (link)). Lungs were cultured for 24 hours in cRPMI (10% FBS, 0.1% 2-mercaptoethanol, 1000U/L Penicillin/Streptomycin, 10–30 μg/mL HDM or 10 μg/mL OVA), and cytokines in lung culture supernatants and BAL fluid (BALF) were quantified using Luminex or ELISA.