1. Inflammation and Apoptosis in Clostridium difficile Enteritis Is Mediated by PGE2 Up- Regulation of Fas Ligand 
Ho Kim, Sang Hoon Rhee, Charalabos Pothoulakis, J. Thomas LaMont 
Gastroenterology 
Volume 133, Issue 3, Pages (September 2007)
DOI: /j.gastro
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2. Figure 1
PGE2 is associated with toxin A-induced colonocyte apoptosis. (A) Human colonocytes (NCM460) grown for 24 hours in the absence of serum were incubated with toxin A (3 nmol/L) and toxin A plus either PGE2 neutralizing antibody (5–10 μg/mL) or NS398 (50–100 μmol/L) for 48 hours. Bars represent mean ± SEM of 3 separate experiments (*P < .005 vs toxin A-stimulated cells). (B) Mice received NS398 (1 mg/kg) intraperitoneally 1 hour prior to formation of ileal loops, which were then exposed to either toxin A (10 μg) or toxin A plus PGE2 (20 μmol/L) for 4 hours. Extracts of ileal scrapings were resolved on 15% polyacrylamide gel and probed with caspase-3 antibody (Data shown are representative of 5 independent samples). Lower panel: Frozen ileal tissues were stained with TUNEL (n = 5/group) (original magnification, ×200). (C) Time course and concentration dependence of PGE2 treatment on colonocyte apoptosis. DNA fragmentation in NCM460 cells exposed to PGE2 was measured by PI staining and FACS analysis. Bars represent mean ± SEM of 3 separate experiments (*P < .001 vs medium-treated cells).
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
PGE2 mediates toxin A-induced FasL expression in colonocytes. (A and B) Colonocytes grown in the absence of serum were incubated with PGE2 (10 μmol/L) or toxin A (3 nmol/L) for the indicated time points. Cell lysates were resolved on polyacrylamide gel and probed with antibodies against FasL, Fas, or β-actin. (C) Colonocytes treated with PGE2 for 48 hours were stained with PE-conjugated FasL antibody and then analyzed by FACS analysis. Results are representative of 3 separate experiments. (D) Colonocytes were incubated with toxin A (3 nmol/L) and toxin A plus either PGE2 blocking antibody (10 μg/mL) or NS398 (100 μmol/L) for 48 hours, and surface FasL expression was determined by FACS analysis. (E) Colonocytes were incubated with toxin A alone, toxin A plus mouse control IgG, and toxin A plus either PGE2 blocking antibody or Fas blocking antibody (5 μg/mL) for 48 hours. (F) Fas blocking antibody inhibits toxin A-induced colonocyte apoptosis. Bars represent mean ± SEM of 3 separate experiments (*P < .005 vs toxin A-stimulated cells).
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
PGE2 induces FasL in vivo. Mice were injected intraperitoneally with NS398 (1 mg/kg) 1 hour prior to formation of ileal loops, which were then exposed to either toxin A or toxin A plus PGE2 for 4 hours. (A) Mucosal extracts were resolved on 10% polyacrylamide gel and probed with the indicated antibodies. Data shown are representative of 5 separate samples. (B) Frozen tissue sections were stained with anti-FasL (green) and -cytokeratin (red) antibodies. Colocalized expression is indicated by yellow in the merged panel (n = 5/group) (original magnification, ×200).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Soluble FasL (sFasL) release from colonocytes exposed to toxin A or PGE2. (A) Colonocytes grown in the absence of serum for 24 hours were exposed to medium, toxin A, or PGE2. Concentrations of human sFasL in medium were measured by ELISA. Data represent mean pg sFasL/mL conditioned medium ± SEM from 3 separate experiments. (B) Colonocytes grown in the absence of serum were exposed to medium, toxin A alone, or toxin A plus either NS398 (100 μmol/L) or PGE2 blocking antibody (10 μg/mL) for 48 hours. Bars represent mean pg sFasL/mL conditioned medium ± SEM from 3 separate experiments (*P < .005 vs toxin A-stimulated cells).
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
FasL on colonocytes induces apoptosis. (A) Six × 105 Jurkat cells per well were coincubated with colonocytes treated with control medium, PGE2, toxin A, or PGE2 plus toxin A for 16 hours. Apoptosis in Jurkat cells was measured by TUNEL assay and assessed by FACS analysis. The percentage of TUNEL-positive cells is indicated. Results are representative of 3 independent experiments. (B) Colonocytes exposed to medium, toxin A, PGE2, or toxin A plus PGE2 for 12 hours were washed and placed in fresh medium for 24 hours. Jurkat cells (in 90 μL of its medium) were incubated with colonocyte conditioned medium (10 μL) containing sFasL, and cell viability was analyzed by MTT assay. (C) Jurkat T cells were incubated with conditioned medium from cultured colonocytes, with or without Fas neutralizing antibody, and then analyzed for cell viability by MTT assay. Jurkat T cells were also incubated with activating Fas antibodies (0.5 μg/mL) for 48 hours as a positive apoptosis control. Bars represent mean ± SEM of 3 separate experiments (*P < .005).
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
The EP1 receptor mediates PGE2-associated FasL induction. (A) Total RNA was isolated from subconfluent colonocytes, and expression levels of EP 1-4 receptors mRNA were determined by RT-PCR using the primers described by Inoue at al.50 Results are representative of 3 independent experiments. (B) Colonocytes were exposed to toxin A alone or toxin A plus either DMSO or NS398 for 24 hours, total RNA was isolated, and mRNA expression of EP receptors was determined by RT-PCR. (C) Colonocytes were incubated with PGE2, 5 μmol/L of the EP1 agonist 17-phenyl trinor PGE2, 10 μmol/L of the EP1/3 agonist sulprostone, 10 μmol/L of the EP2 agonist butaprost, or 10 μmol/L of the EP2/4 agonist 11-deoxy PGE1 for 24 hours. Cell lysates were resolved on 10% polyacrylamide gel and probed with the indicated antibodies. Results shown are representative of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
PGE2/EP1-mediated FasL induction involves activation of NF-κB. (A) Upper: In colonocytes treated with PGE2 for the indicated time points, activation of AKT was analyzed with a phospho-specific antibody (Ser-473). Serum was used as a potent stimulator for the activation of AKT. Lower: Colonocytes were incubated with PGE2, toxin A, or forskolin (FSK; 5 μmol/L), and intracellular cAMP levels were determined by ELISA. Results are representative of 3 independent experiments. (B) Colonocytes were incubated with PGE2 or agonists for EP1 (17-phenyl trinor PGE2) or EP1/3 (sulprostone). Cell lysates were prepared and probed with a phospho-IκB antibody (Ser-32). (C) Nuclear extracts from colonocytes treated with PGE2 or agonists for EP1/3 or EP2/4 (11-deoxy PGE1) were incubated with radiolabelled NF-κB oligo. “Probe” indicates consensus oligo without nuclear extracts. (D) For super shift experiments, rabbit control IgG and p65 antibodies were added before the addition of labeled probe (lanes 3 and 4). Competition assay was performed with unlabelled oligo (cold oligo) (lanes 5 and 6). Results are representative of 3 independent experiments. (E) Colonocytes were stably transfected with the pSuppressorNeo-IKK plasmid expressing IKK-RNAi target sequences or negative control plasmid. Selected control or IKK knockdown cells were incubated with PGE2, and induction of FasL was determined. Results are representative of 3 independent experiments. (F) Subconfluent cultures of control and IKK-silenced cells were incubated with medium, PGE2, or toxin A plus PGE2 for 48 hours, and apoptosis was measured by PI staining and FACS analysis. Bars represent mean ± SEM of 3 separate experiments (*P < .005).
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
B- and T-cell deficiency does not affect toxin A-mediated enteritis. (A and B) Ileal loops of anesthetized control, C3H/HeJ mice, or scid mice were intraluminally injected with PBS or toxin A. After 4 hours, ileal fluid and mucosal scrapings were collected and processed as described below. (A) Fluid secretion was expressed as the loop weight-to-length ratio (mg/cm) (n = 5/group). *P > .5. (B) Concentration of KC in the collected mucosal scrapings. Data are expressed as means ± SEM (n = 5 mice per group, each with triplicate determinations). *P > .1. (C–E) Mice (scid) were preinjected intraperitoneally with FasL (2.5 mg/kg) or PGE2 neutralizing antibodies (5 mg/kg) for 1 hour prior to intraluminal administration of toxin A and processed as described below (n = 5/group). (C) Extracts from ileal mucosal scrapings were resolved on 15% polyacrylamide gels and probed with antibodies against the indicated proteins (Data are representative of 5 independent samples). (D) Ileal fluid secretion (n = 5/group). (E) KC concentration in mucosal scrapings. Data are expressed as means ± SEM (n = 5 mice per group, each with triplicate determinations). *P < .001 vs toxin A-injected mice.
Gastroenterology  , DOI: ( /j.gastro )
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10. Figure 9
Toxin A-induced ileitis in mice with mutated-Fas/FasL system. Ileal loops were prepared in anesthetized control C3H/HeJ mice, FasL−/− (gld), and Fas−/− (lpr) mice and intraluminally injected with PBS, toxin A (3 μmol/L), or toxin A plus PGE2 (20 μmol/L) (n = 5/group). After 4 hours, ileal fluid and ileal mucosal scrapings were collected and processed as described below. (A) PGE2 concentration in ileal fluid. Data are expressed as means ± SEM (n = 5 mice per group, each with triplicate determinations). *P < .005 vs buffer-injected mice. (B) Fluid secretion was expressed as the loop weight-to-length ratio (mg/cm) (n = 5/group, *P < .001 vs toxin A-injected or toxin A plus PGE2-injected control mice). (C and D) One microgram of proteins from the ileal mucosal scrapings was analyzed by ELISA to measure KC and IL-6 concentrations. Data are expressed as means ± SEM (n = 5 mice per group, each with triplicate determinations). *P < .005 vs toxin A-injected or toxin A plus PGE2-injected control mice. (E) Histopathologic scoring for enteric inflammation was evaluated as described previously17 (n = 5/group). #P < .01 vs toxin A-injected control mice. (F) Light micrographs of mouse ileum (H&E staining; original magnification, ×200). (G) The extracts from ileal mucosal scrapings were resolved on 15% polyacrylamide gels and probed with caspase-3 or β-actin antibodies. Data are representative of 5 independent samples. (H) Catalytic action of toxin A in Fas−/− or FasL−/− mice (n = 5). The glucosyltransferase activity of toxin A on RhoA in mucosal scrapings of mice was assessed by Clostridium botulinum C3 exoenzyme ADP-ribosylation assay as described in the Materials and Methods section.
Gastroenterology  , DOI: ( /j.gastro )
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