Adherent invasive Escherichia coli strains from patients with Crohn's disease survive and replicate within macrophages without inducing host cell death

Abstract

Escherichia coli strains recovered from Crohn's disease (CD) lesions are able to adhere to and invade cultured intestinal epithelial cells. We analyzed the behavior within macrophages of adherent invasive E. coli (AIEC) strains isolated from patients with CD. All the 15 AIEC strains tested were able to replicate extensively within J774-A1 cells: the numbers of intracellular bacteria increased 2.2- to 74.2-fold at 48 h over that at 1 h postinfection. By use of murine peritoneal macrophages and human monocyte-derived-macrophages, the reference AIEC strain LF82 was confirmed to be able to survive intracellularly. Transmission electron micrographs of AIEC LF82-infected macrophages showed that at 24 h postinfection, infected cells harbored large vacuoles containing numerous bacteria, as a result of the fusion of several vacuoles occurring after 8 h postinfection. No lactate dehydrogenase (LDH) release, no sign of DNA fragmentation or degradation, and no binding to fluorescein isothlocyanate-labeled annexin V were observed with LF82-infected J774-A1 cells, even after 24 h postinfection. LF82-infected J774-A1 cells secreted 2.7-fold more tumor necrosis factor alpha (TNF-alpha) than cells stimulated with 1 microg of lipopolysaccharide (LPS)/ml. No release of interleukin-1beta was observed with LPS-prestimulated J774-A1 cells infected with AIEC LF82. These findings showed that (i) AIEC strains are able to survive and to replicate within macrophages, (ii) AIEC LF82 replication does not induce any cell death of the infected cells, and (iii) LF82-infected J774-A1 cells release high levels of TNF-alpha. These properties could be related to some features of CD and particularly to granuloma formation, one of the hallmarks of CD lesions.

FIG. 1

Kinetics of intracellular multiplication of AIEC LF82 in J774-AI macrophages. The numbers of viable LF82 bacteria were determined in attached (■) and detached (□) macrophage fractions after different times (hours) of gentamicin treatment. Results are expressed as the number of intracellular bacteria relative to that obtained at 1 h after gentamicin exposure, taken as 100%. Means and standard errors of the means are indicated and correspond to six different experiments.

FIG. 2

Survival of AIEC LF82 (A) and S. enterica serovar Choleraesuis (B) in murine peritoneal macrophages. Means and standard errors of the mean are indicated and correspond to four different experiments in duplicate wells. Bacterial CFU per well containing 5 × 10⁵ macrophages (y axis) and the time after addition of gentamicin (x axis) are shown. Numbers of viable bacteria were determined in the attached (■) and detached (□) macrophage fractions.

FIG. 3

Survival of AIEC LF82 (A) compared to those of serovar Dublin SL2260 (B) and nonpathogenic E. coli K-12 C600 (C) in HMDM. Each time point represents the mean of three independent experiments in triplicate wells. Bacterial CFU per well containing 5 × 10⁵ macrophages (y axis) and the time after addition of gentamicin (x axis) are shown. Numbers of viable bacteria were determined in the attached (■) and detached (□) macrophage fractions.

FIG. 4

DNA electrophoresis of J774-A1 cells at 24 h postinfection. Lanes: L, ladder of molecular size markers in base pairs (SmartLadder SF; Eurogentec); 1, uninfected cells; 2, cells infected with the AIEC strain LF82; 3, cells infected with S. flexneri M90T. Only M90T-infected cells generated ladders of 180 to 200 bp, characteristic of apoptosis. LF82-infected cells were negative for DNA degradation.

FIG. 5

TEMs of J774-A1 macrophages infected at an MOI of 10 bacteria per cell. At 1 h postinfection, AIEC LF82 bacteria were observed in small vacuoles (A). After 8 h of gentamicin treatment, the size of the vacuoles was increased (B). Note phagosome fusion leading to the formation of a large vacuole observed after 24 h of gentamicin treatment (C). Magnification: ×7,200.