Lipocalin 2 is protective against E. coli pneumonia

Abstract

Background: Lipocalin 2 is a bacteriostatic protein that binds the siderophore enterobactin, an iron-chelating molecule produced by Escherichia coli (E. coli) that is required for bacterial growth. Infection of the lungs by E. coli is rare despite a frequent exposure to this commensal bacterium. Lipocalin 2 is an effector molecule of the innate immune system and could therefore play a role in hindering growth of E. coli in the lungs.

Methods: Lipocalin 2 knock-out and wild type mice were infected with two strains of E. coli. The lungs were removed 48 hours post-infection and examined for lipocalin 2 and MMP9 (a myeloid marker protein) by immunohistochemical staining and western blotting. Bacterial numbers were assessed in the lungs of the mice at 2 and 5 days after infection and mortality of the mice was monitored over a five-day period. The effect of administering ferrichrome (an iron source that cannot be bound by lipocalin 2) along with E.coli was also examined.

Results: Intratracheal installation of E. coli in mice resulted in strong induction of lipocalin 2 expression in bronchial epithelium and alveolar type II pneumocytes. Migration of myeloid cells to the site of infection also contributed to an increased lipocalin 2 level in the lungs. Significant higher bacterial numbers were observed in the lungs of lipocalin 2 knock-out mice on days 2 and 5 after infection with E. coli (p < 0.05). In addition, a higher number of E. coli was found in the spleen of surviving lipocalin 2 knock-out mice on day 5 post-infection than in the corresponding wild-type mice (p < 0.05). The protective effect against E. coli infection in wild type mice could be counteracted by the siderophore ferrichrome, indicating that the protective effect of lipocalin 2 depends on its ability to sequester iron.

Conclusions: Lipocalin 2 is important for protection of airways against infection by E. coli.

Figure 1

Lipocalin 2 expression in the lungs of E. coli-infected mice. Immunohistochemical staining using a polyclonal antibody against lipocalin 2 (diluted 1:250) on formalin-fixed lung sections removed 48 hours post-infection with E. coli H9049. Weak staining for lipocalin 2 is found in uninfected bronchial epithelium (A) and alveolear tissue (E) of wild-type C57BL/6 mice. Strong induction is seen following E. coli infection (4 × 10⁷ CFU E. coli H9049/mouse) in wild-type mice (B and F) whereas no staining for lipocalin 2 is seen in infected Lcn2 knock-out mice (C and G). The specificity of the reaction is demonstrated by the lack of staining when using rabbit pre-immune serum (dilution 1:250) as negative control (D and H). Staining for lipocalin 2 was also observed in neutrophils in the bone marrow of wild-type mice (I) but not in Lcn2 knock-out mice (J) or in wild-type mice incubated with pre-immune serum (K).

Figure 2

MMP9 expression in the lungs of E. coli-infected mice. Top. Western blot analysis for lipocalin 2 (Lcn2) and MMP9 (both antibodies diluted 1:1000) of whole lung lysates from uninfected and E. coli-infected (4 × 10⁷ CFU E. coli H9049) wild-type (+/+) and Lcn2 knock-out (-/-) mice. Immunostaining for β-actin (dilution 1:5000) was included to assure equal loading of the samples. Bottom. Immunohistochemical staining for the neutrophil granule protein MMP9 (dilution 1:2000) on formalin-fixed lung sections of wild-type (A-C) and Lcn2 knock-out (D-F) mice. Only a few positive cells were found in the lungs of uninfected mice (A and D) whereas a larger number of cells were stained in the lungs of E. coli infected mice (4 × 10⁷ CFU E. coli H9049/mouse) (B and E). No staining was seen when using a non-specific rabbit Ig as negative control (C and F).

Figure 3

Bacterial numbers in the lungs at 48 hours post-infection. Data are presented as box plots showing CFU/lung (log 10 scale) in wild-type (wt) and Lcn2 knock-out (ko) mice infected with E. coli HB101 or H9049 (both 8 × 10⁷ CFU/mouse) and tested after 48 hours. (A) For E. coli HB101, a statistical significant difference between the CFU in the lungs of wild-type (n = 9) and Lcn2 knock-out mice (n = 8) was observed (p < 0.05). No bacteria were measured in the spleen of these mice. For E. coli H9049, a significant difference was observed for CFU from the lungs (B) (p < 0.01) and spleen (C) (p < 0.05) of wild-type (n = 8) and Lcn2 knock-out (n = 8) mice.

Figure 4

Lcn2 knock-out mice are highly susceptible to lung infection with E. coli H9049. (A) Survival curves of wild-type (wt) (n = 12) and lipocalin 2 knock-out (ko) (n = 16) mice infected with 4 × 10⁷ CFU E. coli H9049/mouse demonstrating a significant higher mortality (p < 0.05) of the knock-out mice. Box plots showing bacterial numbers (CFUs) for the surviving mice (ko (n = 9) and wt (n = 11)) in the lungs (B) and spleens (C). A significantly higher number of bacteria were found in both the lungs (p < 0.05) and spleens (p < 0.05) of Lcn2 knock-out mice than in wild-type mice.

Figure 5

The bacterial load increases in the lungs of mice administered ferrichrome. Bacterial numbers (CFU) after 5 days in the lungs of wild-type mice infected with 4 × 10⁷ CFU E. coli H9049/mouse alone (n = 9) or with 25 mmol desferri-ferrichrome (Des) (n = 10) or iron-loaded ferrichrome (Fer) (n = 13). Three of the mice infected with E. coli H9049 and iron-loaded ferrichrome died and bacterial loads were determined only for the 10 mice surviving to day 5. The number of bacteria in lungs of mice inoculated only with bacteria was significantly lower that in mice also receiving desferri-ferrichrome (p < 0.05) or iron-loaded ferrichrome (p < 0.01). The amount of CFU/lung of mice receiving desferri-ferrichrome + E. coli H9049 was significantly lower than the amount measured in the lungs of the surviving mice that had been administered E. coli H9049 + iron-loaded ferrichrome (p < 0.05). No bacteria were measured in the mice receiving only iron-loaded ferrichrome (n = 6).