Figure 6. Enhanced phagocytosis/autophagy pathways in macrophages by Lcn2.

(a) Protein expression of LC3 and p62 in macrophages at 0, 2, and 4 hours after infection with E. coli. Thioglycollate-elicited peritoneal macrophages were infected with E. coli for 1 hour, and expression of each target protein was evaluated by Western blotting analysis. β-actin is shown as a control. Data shown are representative of each group. (b) Quantification of LC3- II protein in macrophages at 0, 2, and 4 hours after infection with E. coli. Thioglycollate-elicited peritoneal macrophages obtained from Lcn2/IL-10 DKO mice were pre-incubated with or without 100 ng/ml of recombinant Lcn2 for 4 hours before E. coli infection. Expression of LC3 protein was evaluated by Western blotting analysis and is shown as the intensity ratio of LC3- II to LC3- I. N = 6 per group. Error bars represent SEM. *P < 0.05 compared with IL-10 KO mice by one-way ANOVA with Bonferroni’s correction. (c) Ratio of colony forming units (CFU) at the indicated times to CFU at 2 hours after E. coli infection. N = 6 per group. Error bars represent SEM. *P < 0.05 compared with IL-10 KO mice; ^†P < 0.05 compared to Lcn2/IL-10 DKO mice with recombinant Lcn2 pre-treatment by one-way ANOVA with Bonferroni’s correction. (d) Co-localization of Lcn2 and LC3 in macrophages infected with E. coli. Double-immunofluorescent staining with anti-Lcn2 and anti-LC3 antibodies was performed in thioglycollate-elicited peritoneal macrophages obtained from IL-10 KO (upper panels) and Lcn2/IL-10 DKO mice (lower panels). Co-localization of Lcn2 and LC3 is indicated by white arrowheads. Data are representative of each group. Scale bars, 20 μm. DAPI, 4′,6-diamidino-2-phenylindole.