Fig. 2

Absence of Gch1 improves the resistance of mice to in vivo BCG infection. a The excision of the floxed Gch1 allele in both cells from WT and Gch1^fl/flTie2cre recovered by bronchial-alveolar lavage (BAL) was confirmed by genomic PCR, with analysis of BMDM used as a positive control for cre activity: knockout allele (K) floxed allele (F). The presence or absence of the cre transgene was confirmed using HPRT as a housekeeping gene. b The BH4 (pmol/10⁶cells) content of the BAL cells was analysed by HPLC (n = 4 per genotype, two-tailed t-test, p < 0.05). c, d Gch1^fl/flTie2cre (red) and wildtype (black) mice were infected intranasally with 5 × 10⁶ colony forming units (CFU) BCG Pasteur. Four weeks following infection the lungs and spleen were removed and homogenised for analysis along with tissues from a matched cohort of uninfected animals. The bacterial load of the organs was enumerated by CFU count from lung (c) and spleen (d). Each dot represents one animal, line represents median CFU of each group (n = 10 Gch1^fl/fl, n = 6 Gch1^fl/flTie2cre (Spleen), n = 7 Gch1^fl/flTie2cre (Lung) per group), p < 0.01 (Mann–Whitney test). e Spleen to body weight ratio was also calculated. Remaining tissue homogenate was used to measure total biopterin content, in lungs (f) and spleen (g) (n = 10 Gch1^fl/fl, n = 7 Gch1^fl/flTie2cre Spleen (infected) and n = 4/genotype (uninfected), p < 0.05). Total lung biopterin content was correlated to lung CFU in all animals. Pearson r p < 0.05 (h). i iNOS protein expression was confirmed by western blotting, with ß-tubulin loading control (n = 2 per group shown, representative of n = 3 independent biological replicates assayed in total). An NO analyzer was used to measure NOx content in the lung homogenates (p < 0.05) (j). All data analsyed with ANOVA with Bonferroni post-test (apart from (b–d) and (h) as above). Data shown as individual data-points with mean and SEM (apart from (c) and (d) as above). *p < 0.05. Source gel and blot images are available in Supplementary Figure 10