Catalase

Streptococcus pneumoniae (S. pneumoniae) isolate serotype 23F (neomycin^R) and a previously described isogenic S. pneumoniae pgdA mutant (kanamycin^R and neomycin^R) were used throughout the study^{21 (link)}. For mouse infections, all pneumococcal strains were grown in tryptic soy (TS) broth (BD) at 37°C without aeration to an optical density of 1.0 at 620 nm. For in vivo bacterial colonization, pneumococci were incubated on TS plates supplemented with 100μl of catalase (30,000 U/ml; Worthington Biomedical) and either 5μg/ml neomycin (FisherScientific) or 125μg/ml kanamycin (Sigma) at 37°C in 5% CO₂ overnight. Staphylococcus aureus (S. aureus) strain USA300-JE2 and S. aureus oatA::bursa USA300-JE2 (erythromycin^R) were used for infant mouse oral infections. All S. aureus strains were grown overnight, with shaking, at 37°C in 5ml of TS broth, diluted 100-fold next day and subcultured for 4 hours in 5ml of TS broth. For in vivo bacterial colonization S. aureus strains were plated on CHROMID MRSA SMART II agar (bioMérieux), TS or TS+5μg/ml erythromycin and incubated overnight at 37°C.

Fetal PASMC from normal and CDH lambs were plated on a 6 well tissue culture plate at 1×10⁶ cells/well in DMEM with 10% FBS and allowed to adhere overnight. On day 0, media was changed to DMEM with 2.5% FBS (as this was the lowest concentration that supported growth). On day 4 cells were removed from the wells using 0.25% trypsin/0.53mM ethylene-diaminetetraacetic acid (EDTA) digestion and counted using a hemocytometer. Absolute cell number on day 4 was compared to cell number at day 0 to determine the relative increase over time. Comparisons were made between control and CDH cells. This assay was repeated twice in the presence or absence of SOD (250U/ml; Sigma Aldrich, St Louis, MO) plus catalase (26.3U/ml; Worthington Biochemical) and Bosentan (a non-selective ET A and B receptor inhibitor) (1mM). Preliminary experiments evaluating the effects of SOD or catalase alone were performed, which revealed that either alone had no effect on either PAEC or PASMC growth (data not shown). For this reason, all experiments were performed with SOD plus catalase in combination.

All bacterial strains were animal passaged prior to use in experiments and stored at −80°C in 20% glycerol. Inocula consisted of 10(x005E)7 mid-log-phase PBS-washed bacteria in 10μl PBS and were plated to confirm dose. They were delivered to the nares of unanesthetized mice as previously described (37 (link)). At the indicated time points, mice were sacrificed, their trachea cannulated, and 200μl PBS instilled. Lavage fluid was collected from the nares and serially diluted in PBS for plating on TS agar plates supplemented with catalase (Worthington Biochemicals). The lower limit of detection was either 100 CFU/ml or 20 CFU/ml lavage fluid, depending on the experiment. Blood was collected by cardiac puncture and the serum separated. Serum and lavages were assayed for mMIF by specific ELSIA. Serum was additionally assayed for anti-pneumococcal IgG as previously described (14 (link)).
Recombinant murine MIF (rMIF) was produced as described previously and ensured to be LPS-free (38 (link)). PBS was used to dilute the rMIF to the indicated concentration. As dimethyl sulfoxide (DMSO) was present in the rMIF preparation, an identical amount of DMSO was added to PBS for the vehicle control treatment. Either rMIF or vehicle control were administered in a 10μl volume to the nares of unanesthetized mice for the frequency and duration described.