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. 2004 Mar;164(3):947-57.
doi: 10.1016/S0002-9440(10)63182-3.

Epithelia under metabolic stress perceive commensal bacteria as a threat

Aisha Nazli  1 , Ping-Chang Yang, Jennifer Jury, Kathryn Howe, James L Watson, Johan D Söderholm, Philip M Sherman, Mary H Perdue, Derek M McKay
Affiliations

Epithelia under metabolic stress perceive commensal bacteria as a threat

Aisha Nazli et al. Am J Pathol. 2004 Mar.

Abstract

The normal gut flora has been implicated in the pathophysiology of inflammatory bowel disease and there is increased interest in the role that stress can play in gut disease. The chemical stressor dinitrophenol (DNP, uncouples oxidative phosphorylation) was injected into the ileum of laparotomized rats and mitochondria structure, epithelial permeability, and inflammatory cell infiltrate were examined 6 and 24 hours later. Monolayers of human colonic epithelial cells (T84, HT-29) were treated with DNP +/- commensal Escherichia coli, followed by assessment of epithelial permeability, bacterial translocation, and chemokine (ie, interleukin-8) synthesis. Delivery of DNP into rat distal ileum resulted in disruption of epithelial mitochondria; similar changes were noted in mildly inflamed ileal resections from patients with Crohn's disease. Also, DNP-treated ileum displayed increased gut permeability and immune cell recruitment. Subsequent studies revealed deceased barrier function, increased bacterial translocation, increased production of interleukin-8, and enhanced mobilization of the transcription factor AP-1 in the model epithelial cell lines exposed to commensal bacteria (E. coli strains HB101 or C25), but only when the monolayers were pretreated with DNP (0.1 mmol/L). These data suggest that enteric epithelia under metabolic stress perceive a normally innocuous bacterium as threatening, resulting in loss of barrier function, increased penetration of bacteria into the mucosa, and increased chemokine synthesis. Such responses could precipitate an inflammatory episode and contribute to existing enteric inflammatory disorders.

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Figures

Figure 1
Figure 1
Mildly inflamed ileum from patients with Crohn’s disease has abnormal mitochondria. Transmission EM reveals swollen mitochondria with irregular cristae in enterocytes from patients with Crohn’s disease (arrows) (a) that were not apparent in ileal resections from patients with cancer (b) (n = 3). Scale bars, 2 μm.
Figure 2
Figure 2
DNP treatment results in mitochondrial abnormalities, increased permeability, and an inflammatory infiltrate in the rat ileum. Tissues from controls show numerous electron-dense mitochondria (A), whereas those in tissues from ileum exposed to DNP 6 hours previously have swollen disrupted mitochondria (arrowheads) (B). C: Bacteria (arrowheads) were commonly observed within enterocytes of tissue from DNP-treated rats. Tissue ion conductance (D) and flux of HRP (E) were increased in ileal segments from DNP-treated rats at 6 hours and 24 hours after treatment. F: Concomitantly these tissues showed evidence of an inflammatory infiltrate as assessed by mononuclear cell and granulocyte (neutrophil, eosinophil, and mast cells) counts on H&E-stained sections (n = 4; *, P < 0.05 compared to PBS/dimethyl sulfoxide control; mean ± SEM). Scale bars, 2 μm.
Figure 3
Figure 3
Loss of barrier function occurs in metabolically stressed polarized T84 epithelia exposed to nonpathogenic E. coli strains. A: Monolayers treated with DNP + E. coli HB101 display a drop in TER 10 hours later (n = 3; mean ± SD; *, P < 0.05 compared to other treatments). B: Bar chart showing that 24 hours of exposure to DNP (0.1 mmol/L) + E. coli HB101 (HB101; 106 cfu) reduces TER (presented as percent of pretreatment values of the same monolayer) and that while E. coli C25 (C25) alone reduces TER, this is enhanced in the presence of DNP. The effect of EPEC is included as a positive control (n = 9; *, P < 0.05 compared to control; #, P < 0.05 compared to C25 only). C: Under the same conditions, DNP + HB101 exposure evoked increases in transepithelial flux of HRP (n = 6; *, P < 0.05 compared to control). D: A 30- to 60-minute pretreatment with an ERK MAPK inhibitor (i) (U0126; 25 μmol/L; n = 4), or inhibitors of PI-3K (LY294002; 20 μmol/L; n = 4) or MLCK (ML-7; 20 μmol/L; n = 4) did not prevent the DNP + E. coli HB101-induced drop in TER observed after a 24-hour culture. As a control, the MCLKi partially reduced the EPEC-induced drop in TER (*, P < 0.05 compared to control; #, P < 0.05 compared to EPEC only; n = 4; mean ± SEM). In these experiments pretreatment TER ranged from 1000 to 2500 Ω · cm2. E: En face confocal scanning laser microscope images of epithelial F-actin (left) and α-actinin (right) showing that 24 hours of DNP + E. coli HB101 co-treatment resulted in disruption of the normal enterocytic cytoskeleton as shown by focal condensation of F-actin (arrows) and dissociation from the cell periphery/perijunctional region (*), and redistribution and focal hot spots of α-actinin (arrows) (images are representative of six monolayers/condition).
Figure 4
Figure 4
Bar chart showing the significant drop in TER across T84 epithelial monolayers 24 hours after treatment with indomethacin (indo.; 10 μmol/L) + E. coli HB101 (n = 11 monolayers; * and **, P < 0.05 compared to other treatments; note in these experiments HB101 alone resulted in a small but significant drop in TER; pretreatment TER ranged from 1380 to 3760 Ω · cm2).
Figure 5
Figure 5
Exposure to DNP + E. coli HB101 results in increased bacterial entry and translocation across polarized epithelial monolayers. a and b: Representative EM photomicrographs showing bacteria within T84 cells (arrows) and in the paracellular spaces (arrowheads) between adjacent DNP + E. coli HB101-treated filter-grown T84 epithelial monolayers. c: Using a modified invasion assay, ∼10-fold more bacteria were obtained from cell lysates of DNP + E. coli HB101-treated cells compared to those exposed to E. coli only (data are expressed as the percentage of the number of extracellular bacteria 24 hours after culture; n = 18; *, P < 0.05 compared to control). Scale bars, 1 μm.
Figure 6
Figure 6
Metabolic stress and exposure to nonpathogenic E. coli evokes increased IL-8 production and AP-1 activation. A: Image representative of three separate experiments showing increased IL-8 mRNA reverse transcriptase-polymerase chain reaction product in epithelia treated with DNP + E. coli, which is semiquantified by densitometry (average of the two separate epithelial preparations shown in A). B and C: Bar chart showing that increased HT-29 IL-8 production is blocked by use of pharmacological inhibitors of ERK and p38 MAPKs, and NF-κB (n = 5; *, P < 0.05 compared to control, E. coli HB101 or DNP only). D: Representative electrophoretic mobility shift assay gel showing time-dependent induction in AP-1 activation in DNP + E. coli HB101-treated epithelia (left). Specificity of the AP-1 band was confirmed by ablation of the band by inclusion of a cold competitor (c.c.) double-stranded oligonucleotide but not an irrelevant cold competitor (irr. c.c.) and supershifting by a pan-anti-Fos antibody (Ab), but not an irrelevant isotype-matched antibody (irr. Ab.) (right; extracts from cells 2 hours after DNP + E. coli HB101 treatment; NS, nonspecific band; FP, free probe). Extracts from TNF-α (10 ng/ml)-treated epithelia were used as a positive control (not shown).
Figure 7
Figure 7
Hypothetical model for the interplay of metabolically perturbed gut epithelia and the normal gut flora in the induction of intestinal inflammatory disease and/or disease relapse.
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