Figure 10. Splenic Fpn1 expression, Nrf2 activity, and DFX-treatment in systemic S. typhimurium infection. (A–D) Nos2+/+ and Nos2−/− mice were infected i.p. with S. typhimurium (S. tm.) and euthanized after 24 or 72 h, respectively. (A) Bacterial loads in splenic lysates were determined and depicted as colony forming units after log transformation (logCFU; n = 10, 12, 5, and 11 mice, respectively). (B) Splenic samples of mice infected with Salmonella for 72 h were used for protein preparation and Fpn1 expression was analyzed by Western blotting. Actin is shown as loading control. Data from one of four independent experiments are depicted. (C) Fpn1 and actin levels were quantified by densitometric scanning of membranes. Data were compared by ANOVA using Bonferroni’s correction (n = 8 independent experiments). (D) The binding affinity of Nrf2 in spleens of Nos2+/+ and Nos2−/− mice was determined in nuclear extracts as described after infection with Salmonella for 72 h. Data were compared and depicted as in Fig. 2 A (n = 6, 8, 8, and 10 mice, respectively). (E–I) In independent experiments, Nos2+/+ and Nos2−/− mice were i.p. infected with wild-type S. typhimurium (S. tm. WT) or an isogenic triple mutant, deficient in enterobactin synthesis, sitABCD-, and feo-mediated iron uptake (S. tm. entC sit feo). Salmonella wild-type–infected mice were randomized in two groups to receive either DFX dissolved in drinking water or the same volume of drinking water (Solv.). Bacterial loads (E; n = 8, 13, 13, 7, 8, and 11 mice, respectively) in spleens were quantified and TNF (F; n = 8, 13, 7, and 8 mice, respectively) expression were analyzed by qRT-PCR normalized to the housekeeping gene Hprt. (G-I) Prussian blue staining. Bar, 200 µm. (J) Hepatic hepcidin mRNA expression was determined by qRT-PCR and normalized for Hprt levels. Data were compared by ANOVA using Bonferroni’s correction (n = 9, 10, 12, 6, 5, and 11 mice, respectively). (K) Serum iron levels were colorimetrically measured and compared by ANOVA using Bonferroni’s correction (n = 9, 10, 12, 6, 5 and 11 mice, respectively). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 10.

Splenic Fpn1 expression, Nrf2 activity, and DFX-treatment in systemic S. typhimurium infection. (A–D) Nos2+/+ and Nos2−/− mice were infected i.p. with S. typhimurium (S. tm.) and euthanized after 24 or 72 h, respectively. (A) Bacterial loads in splenic lysates were determined and depicted as colony forming units after log transformation (logCFU; n = 10, 12, 5, and 11 mice, respectively). (B) Splenic samples of mice infected with Salmonella for 72 h were used for protein preparation and Fpn1 expression was analyzed by Western blotting. Actin is shown as loading control. Data from one of four independent experiments are depicted. (C) Fpn1 and actin levels were quantified by densitometric scanning of membranes. Data were compared by ANOVA using Bonferroni’s correction (n = 8 independent experiments). (D) The binding affinity of Nrf2 in spleens of Nos2+/+ and Nos2−/− mice was determined in nuclear extracts as described after infection with Salmonella for 72 h. Data were compared and depicted as in Fig. 2 A (n = 6, 8, 8, and 10 mice, respectively). (E–I) In independent experiments, Nos2+/+ and Nos2−/− mice were i.p. infected with wild-type S. typhimurium (S. tm. WT) or an isogenic triple mutant, deficient in enterobactin synthesis, sitABCD-, and feo-mediated iron uptake (S. tm. entC sit feo). Salmonella wild-type–infected mice were randomized in two groups to receive either DFX dissolved in drinking water or the same volume of drinking water (Solv.). Bacterial loads (E; n = 8, 13, 13, 7, 8, and 11 mice, respectively) in spleens were quantified and TNF (F; n = 8, 13, 7, and 8 mice, respectively) expression were analyzed by qRT-PCR normalized to the housekeeping gene Hprt. (G-I) Prussian blue staining. Bar, 200 µm. (J) Hepatic hepcidin mRNA expression was determined by qRT-PCR and normalized for Hprt levels. Data were compared by ANOVA using Bonferroni’s correction (n = 9, 10, 12, 6, 5, and 11 mice, respectively). (K) Serum iron levels were colorimetrically measured and compared by ANOVA using Bonferroni’s correction (n = 9, 10, 12, 6, 5 and 11 mice, respectively). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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