Crohn's disease-associated Escherichia coli survive in macrophages by suppressing NFκB signaling

Abstract

Background: Epidemiological and genetic studies suggest a role for enteric flora in the pathogenesis of Crohn's disease (CD). Crohn's disease-associated Escherichia coli (CDEC) is characterized by their ability to invade epithelial cells and survive and induce high concentration of TNF-α from infected macrophages. However, the molecular mechanisms of CDEC survival in infected macrophages are not completely understood.

Methods: Intracellular survival of CDEC strain LF82 isolated from inflamed ileum tissue, 13I isolated from inflamed colonic tissue, and control E. coli strains were tested in the murine macrophage cell line, J774A.1 by Gentamicin protection assay. Modulation of intracellular cell signaling pathways by the E. coli strains were assessed by western blot analysis and confocal microscopy.

Results: 13I demonstrated increased survival in macrophages with 2.6-fold higher intracellular bacteria compared with LF82, yet both strains induced comparable levels of TNF-α. LF82 and 13I differentially modulated key mitogen-activated protein kinase pathways during the acute phase of infection; LF82 activated all 3 mitogen-activated protein kinase pathways, whereas 13I activated ERK1/2 pathway but not p38 and JNK pathways. Both 13I and LF82 suppressed nuclear translocation of NFκB compared with noninvasive E. coli strains during the acute phase of infection. However, unlike noninvasive E. coli strains, 13I and LF82 infection resulted in chronic activation of NFκB during the later phase of infection.

Conclusions: Our results showed that CDEC survive in macrophages by initially suppressing NFκB activation. However, persistence of bacterial within macrophages induces chronic activation of NFκB, which correlates with increased TNF-α secretion from infected macrophages.

Fig. 1. Crohn's disease-associated Escherichia coli (CDEC) strain, 13I invades and replicates in macrophages and epithelial cells

(A) J774.A1 macrophages and (B) Caco2 cells were infected for 3 hrs with CD-associated E. coli strains LF82 and 13I, and intracellular CFU was measured by Gentamicin protection assay, and expressed as percentage of initial inoculum (I/O). Non-pathogenic E coli strain, EFC-1 and UC-associated E coli strain, 30A were used as controls. Data are mean ± S.D. Statistical significance was determined by student's t test.

Fig. 2. Crohn's disease-associated Escherichia coli (CDEC) infected macrophages secrete high level of TNFα

Cytokines secreted by J774.A1 macrophages infected with E. coli strains were measured by ELISA at 24 hrs post infection. (A) TNFα (B) IL-6 (C) IL-10. Data are mean ± S.D. Statistical significance was determined by student's t test.

Fig. 3. Crohn's disease-associated Escherichia coli (CDEC) strain, 13I activates ERK1/2 but not p38 and JNK pathways

(A, C, E) Representative western blots of cell lysates from J774.A1 macrophages infected with E. coli strains for 30 min or 1 hr and (B, D, F) densitometric analyses of the western blots of three independent experiments. Data are mean ± S.D. Statistical significance was determined by student's t test.

Fig. 4. Crohn's disease-associated Escherichia coli (CDEC) suppress activation and nuclear translocation of NFκB during the initial phase of infection

(A, C) J774.A1 macrophages were infected with E. coli strains for (A) 30 min or (C) 1 hr and cell lysates were analyzed by western blotting. The western blots are representative of three independent experiments and the histograms represent densitometric analyses of western blots from two independent experiments. (B, D) Macrophages grown on coverslips were infected with E. coli strains for (B) 30 min or (D) 1 hr and were subjected to confocal imaging. Scale 10 µM.

Fig. 5. Intracellular replication of Crohn's disease-associated Escherichia coli (CDEC) results in activation of NFκB during the chronic phase of infection

(A) J774.A1 macrophages grown on coverslips were infected with E. coli strains for 3 hr. After 3 hrs, cells were washed and again incubated for 3 hrs in complete medium supplemented with 10% serum and 50 ug/mL Gentamicin. Scale 10 µM.