Wild type mice

Wild-type (WT) mice on the C57BL/6J background were obtained from Jackson Lab. Cdh5-Cre^ERT2 mice were generated by Ralf Adams (Max Planck) and kindly provided by Nancy Speck (University of Pennsylvania) and Bisen Ding (Cornell)^{67 (link)}. Il6^fl/fl mice have been described previously^{68 (link), 69 (link)}. Cdh5-Cre^ERT2;Il6^fl/fl mice were generated by crossing Il6^fl/fl mice with Cdh5-Cre^ERT2 mice. Mice were genotyped with primers including IL-6 FP: 5′-CCCACCAAGAACGATAGTCA-3′, and IL-6 RP: 5′-GGTATCCTCTGTGAAGTCCTC-3′. Cdh5-Cre^ERT2;Il6^fl/fl and Il6^fl/fl mice (2 weeks old) were intraperitoneally injected with 0.1 ml of 5 mg/ml tamoxifen daily for consecutive 5 days. Rosa-LSL-tdTomato;Tie2-Cre mice were generated by crossing Rosa-LSL-tdTomato mice (Jackson Lab) with Tie2-Cre mice (Jackson Lab). All animal studies were reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) at the University of Pennsylvania. All animals were housed in the Association for the Assessment and Accreditation of Laboratory Animal Care-accredited animal facility of the University of Pennsylvania.

Mice with a targeted mutation in Twsg1 (Twsg1^{tm1.1 Mboc}) were as described previously (Petryk et al., 2004 (link)). Wild-type (WT) mice were purchased from Jackson Laboratories. All mice were of the C57BL/6 background. Presence of a spermatic plug was counted as embryonic day 0.5 (E0.5). Pregnant females were treated by gavage with all-trans retinoic acid (ATRA, Sigma, St Louis, MO) in corn oil at doses of 3.75 or 7.5 mg/kg of body weight (Kotch et al., 1995 (link)) on the morning (10 am) of E7.5, which is a well defined teratogenic window for the induction of HPE (Higashiyama et al., 2007 (link); Lipinski et al., 2010 (link)). Subsequently the pregnant females were killed by CO₂ inhalation, embryos were isolated at E9.5 or E10.5, and assessed for external phenotypes under the dissecting microscope, including telencephalic vesicle abnormalities consistent with HPE and neural tube defects. For geometric morphometric shape analysis, embryos were collected at E11.5 and fixed in 4% paraformaldehyde with glutaraldehyde (Schmidt et al., 2010 (link)). Mice were housed in specific pathogen-free (SPF) conditions. Standard chow and water were provided ad libitum. All animal procedures were approved by the University of Minnesota Institutional Animal Care and Use Committee.

4–6-week IFN-γ  −/− knockout (KO), IFN-γ receptor −/− KO, and +/+ wild-type (WT) mice (all on a C57BL/6 background) were obtained from the Jackson Laboratory (Bar Harbor, ME) and kept in a pathogen-free environment.

SP-A humanized transgenic mice were generated as previously described (15 (link)). SP-A^-/- mice on C57BL/6 background (16 (link)) were bred in-house and wild-type (WT) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and bred in house for experiments. Age-matched (approximately 6-8 weeks) male mice were used for experiments since they have more robust methacholine sensitivity for lung function measurements compared to female mice. Mice were anesthetized under inhaled isoflurane and given 3.9 μg of recombinant IL-13 (Peprotech) in 50 μl of sterile saline via oropharyngeal delivery (17 (link)). At the desired time point, mice were euthanized and the lungs lavaged with PBS (0.1 mM EDTA) and lung tissue was obtained for further analysis. Differential cell counts were analyzed from the lavage fluid after H&E staining. Viability was assessed by Trypan blue exclusion.

Studies were approved by the Penn State University and University of New Mexico Institutional Animal Care and Use Committee. TLR-2 null (TLR-2−/−) and wild-type (WT) mice (both of C57BL/6 background) of 9–12 weeks of age were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). BB effect on intestinal permeability in an in-vivo mouse model system was determined using a recycling intestinal perfusion method [1 (link)]. For in vivo studies, 1 × 10⁹ CFU of BB in 200 μL PBS were administered daily by oral-gastric gavage and mouse intestinal permeability was measured at different treatment periods. A 6–8 cm segment of mouse small intestine was isolated and cannulated with a small diameter plastic tube (in an anesthetized mouse) and continuously perfused with 5 mL Krebs-phosphate saline buffer for a 2 h perfusion period. An external recirculating pump was used to recirculate the perfusate at a constant flow rate (0.75 mL/min). The intestinal permeability was assessed by measuring luminal-to-serosal flux rate of paracellular probe, Texas Red-labeled dextran (MW = 10,000 g/mol) [27 (link),71 (link)].