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Host Response and Inflammation

Adherent Invasive Escherichia coli Strains from Patients with Crohn's Disease Survive and Replicate within Macrophages without Inducing Host Cell Death

Anne-Lise Glasser, Jerome Boudeau, Nicolas Barnich, Marie-Helene Perruchot, Jean-Frederic Colombel, Arlette Darfeuille-Michaud
Anne-Lise Glasser
Pathogénie Bactérienne Intestinale, Laboratoire de Bactériologie, Universitéd'Auvergne, 63001 Clermont-Ferrand, and
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Jerome Boudeau
Pathogénie Bactérienne Intestinale, Laboratoire de Bactériologie, Universitéd'Auvergne, 63001 Clermont-Ferrand, and
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Nicolas Barnich
Pathogénie Bactérienne Intestinale, Laboratoire de Bactériologie, Universitéd'Auvergne, 63001 Clermont-Ferrand, and
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Marie-Helene Perruchot
Pathogénie Bactérienne Intestinale, Laboratoire de Bactériologie, Universitéd'Auvergne, 63001 Clermont-Ferrand, and
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Jean-Frederic Colombel
Laboratoire de Recherche sur les Maladies Inflammatoires de l'Intestin, Centre Hospitalier Universitaire, 59000 Lille, France
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Arlette Darfeuille-Michaud
Pathogénie Bactérienne Intestinale, Laboratoire de Bactériologie, Universitéd'Auvergne, 63001 Clermont-Ferrand, and
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DOI: 10.1128/IAI.69.9.5529-5537.2001
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Figures

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  • Fig. 1.
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    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.
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    Fig. 2.

    Survival of AIEC LF82 (A) and S. entericaserovar 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 (yaxis) and the time after addition of gentamicin (xaxis) are shown. Numbers of viable bacteria were determined in the attached (■) and detached (□) macrophage fractions.

  • Fig. 3.
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    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.
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    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.
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    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.

Tables

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  • Table 1.

    Uptake and ability of E. coli strains isolated from patients with CD to evade killing by J774-A1 cells after 1, 4, 8, 24, and 48 h of gentamicin treatment

    MOI and strainUptake of bacteria (104CFU)a after 1 h of treatment% Survival of bacteriab after the indicated treatment period
    4 h8 h24 h48 h
    MOI = 10
     K-12 C6002.4 ± 0.2 122 ± 5124 ± 11 6 ± 1 0.6 ± 0.1
     LF8212.6 ± 1.1 217 ± 31 280 ± 40 504 ± 631122 ± 297
     LF9 32.5 ± 20.3 132 ± 22201 ± 85 969 ± 261 1743 ± 798
     LF1517.9 ± 6.7 74 ± 7 98 ± 6 624 ± 212700 ± 57
     LF16 18.5 ± 4.8 162 ± 25205 ± 20 3537 ± 418 7421 ± 1246
     LF288.5 ± 2.2 213 ± 92 212 ± 62 563 ± 120291 ± 85
     LF31 8.1 ± 3.6 130 ± 6146 ± 21 850 ± 137 1676 ± 317
     LF4315.1 ± 7.5 258 ± 71 455 ± 124 820 ± 164910 ± 319
     LF46 0.5 ± 0.04 364 ± 160670 ± 221 2782 ± 582 4286 ± 633
     LF507.5 ± 1.0 156 ± 19 346 ± 13 2071 ± 4905506 ± 417
     LF65 12.1 ± 2.1 286 ± 42423 ± 81 1239 ± 31 3267 ± 44
     LF714.8 ± 0.6 259 ± 28 600 ± 213 646 ± 278782 ± 277
     LF100 13.8 ± 2.9 191 ± 16248 ± 46 251 ± 47 265 ± 24
     LF10633.1 ± 1.0 156 ± 24 184 ± 18 365 ± 169225 ± 106
     LF107 2.2 ± 0.8 147 ± 11420 ± 186 572 ± 237 591 ± 212
     LF12010.5 ± 1.2 158 ± 21 167 ± 67 495 ± 1371451 ± 274
    MOI = 10
     K-12 C600 6.7 ± 1.8 145 ± 4 ND2.18 ± 1.10 0.8 ± 0.1
     S. entericaserovar Chloeraesuis 253.0 ± 61.2 134 ± 29 ND0.04 ± 0.02 ND
     S. flexneri M90T 10.4 ± 1.2 57 ± 11 ND0.40 ± 0.30 ND
     LF82 75.0 ± 26.0 237 ± 41268 ± 84 450 ± 59851 ± 264
    • ↵a Numbers of CFU recovered from lysed monolayers; means ± standard errors of the means determined from three to five independent experiments in duplicate.

    • ↵b Mean percentages ± standard errors of the means of intracellular bacteria relative to the number after 1 h of gentamicin treatment, defined as 100%. ND, not determined.

  • Table 2.

    Absence of LDH release by J774-A1 cells infected with AIEC strain LF82

    Gentamicin treatment period (h)LDH activitya (% cytotoxicityb) at an MOI of:
    10100
    AIEC LF82K-12 C600S. flexneri M90TSerovar CholeraesuisAIEC LF82K-12 C600S. flexneri M90TSerovar Choleraesuis
    1 20.8 ± 4.7 (2.2)16.1 ± 4.2 (0.4)  43.4 ± 15.6 (10.8)  27.0 ± 7.0 (4.6) 19.8 ± 4.8 (1.9) 21.7 ± 4.4 (2.6) 82.8 ± 19.2 (25.7)  29.5 ± 4.5 (5.5)
    422.2 ± 5.2 (2.8) 23.2 ± 3.1 (3.2)  83.8 ± 37.1 (26.8)  31.0 ± 1.0 (6.1) 19.8 ± 5.2 (1.9)23.0 ± 5.7 (3.1) 131.5 ± 32.0 (44.2)  56.5 ± 2.5 (15.89)
    8 29.5 ± 4.5 (5.5) 24.5 ± 9.5 (3.6)177.0 ± 29.5 (61.6)  35.5 ± 13.5 (7.8) 26.0 ± 8.0 (4.2) 27.5 ± 2.5 (4.8) 202.0 ± 5.0 (70.9)125.5 ± 61.5 (41.9)
    24 41.8 ± 5.3 (10.2)43.8 ± 9.6 (10.9) ND 148.5 ± 0.4 (50.6) 42.8 ± 3.8 (10.6) 42.3 ± 4.4 (10.4)ND 208.5 ± 11.5 (73.3)
    • ↵a Determined in supernatants by using NADH as a substrate. Release of LDH was measured at different gentamicin treatment times after a 2-h infection period and expressed as units per liter of supernatant (means ± standard errors of the means n = 3 independent experiments in duplicate). ND, not determined.

    • ↵b Calculated as (experimental release − spontaneous LDH release at 1 h)/(total LDH release at 1 h − spontaneous release at 1 h) × 100. Spontaneous LDH release by uninfected cells were 14.9 ± 4.2 and 43.2 ± 5.2 after 1 and 24 h of gentamicin treatment, respectively.

  • Table 3.

    Enumeration of apoptotic and necrotic J774-A1 macrophages after bacterial infection or treatment with gliotoxin

    % Apoptotic cellsa% Necrotic cellsa
    8 h postinfection24 h postinfection8 h postinfection24 h postinfection
    Untreated cells 10.1 ± 0.911.2 ± 1.2 1.6 ± 1.1 2.0 ± 0.7
    AIEC LF827.8 ± 0.2 10.0 ± 1.6 1.4 ± 0.6 4.0 ± 1.4
    Gliotoxin (5 μM) 36.6 ± 1.0 ND4.5 ± 0.4 ND
    S. flexneriM90T 26.6 ± 2.8 19.5 ± 4.0 5.0 ± 1.212.4 ± 1.8
    Serovar Cholereasuis 25.0 ± 2.328.0 ± 0.8 2.0 ± 1.2 5.5 ± 2.6
    K-12 C60010.9 ± 2.5 11.7 ± 0.4 1.1 ± 1.10.9 ± 0.0
    • ↵a Mean percentages ± standard errors of the means of apoptotic or necrotic cells visualized by using the annexin V-FITC assay (Sigma). The percentage of stained cells was scored by analysis of 100 cells of three independent experiments in duplicate. ND, not determined.

  • Table 4.

    Production of proinflammatory cytokines IL-1β and TNF-α by J774-A1 cells after AIEC LF82 infection

    Gentamicin treatment period (h)IL-1β concn (pg/ml)ain:TNF-α concn (pg/ml)b
    Uninfected cellsUninfected lysed cellsAIEC LF82- infected cellsS. flexneri M90T infected cellsUninfected cellsAIEC LF82- infected cellsLPS-stimulated cells
    1 <10 (0%)97.0 ± 2.8 (100%) <10 (0%)40.5 ± 0.7 (41.7%)214 ± 48 99 ± 6 110 ± 5
    24 <10 (0%) ND <10 (0%) ND 302 ± 133,533 ± 111 1,300 ± 86
    • ↵a J774-A1 cells were prestimulated with LPS and then infected with S. flexneri M90T or AIEC LF82. Medium containing gentamicin was added, and IL-1β concentrations in culture supernatant were measured by ELISA at the indicated times of gentamicin treatment. Results are means ± standard deviations from two independent experiments performed in duplicate, expressed in picograms per milliliter and as percentage of total IL-1β. ND, not determined.

    • ↵b J774-A1 cells were exposed to AIEC LF82 or to LPS for 2 h. Medium containing gentamicin was added, and TNF-α concentrations in culture supernatant were measured by ELISA at the indicated times of gentamicin treatment. Results are means ± standard errors of the means of two independent experiments performed in duplicate, expressed in picograms of secreted TNF-α per milliliter.

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Adherent Invasive Escherichia coli Strains from Patients with Crohn's Disease Survive and Replicate within Macrophages without Inducing Host Cell Death
Anne-Lise Glasser, Jerome Boudeau, Nicolas Barnich, Marie-Helene Perruchot, Jean-Frederic Colombel, Arlette Darfeuille-Michaud
Infection and Immunity Sep 2001, 69 (9) 5529-5537; DOI: 10.1128/IAI.69.9.5529-5537.2001

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Adherent Invasive Escherichia coli Strains from Patients with Crohn's Disease Survive and Replicate within Macrophages without Inducing Host Cell Death
Anne-Lise Glasser, Jerome Boudeau, Nicolas Barnich, Marie-Helene Perruchot, Jean-Frederic Colombel, Arlette Darfeuille-Michaud
Infection and Immunity Sep 2001, 69 (9) 5529-5537; DOI: 10.1128/IAI.69.9.5529-5537.2001
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KEYWORDS

Bacterial Adhesion
Crohn Disease
Escherichia coli
macrophages

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