Cyclooxygenase-2 deficiency leads to intestinal barrier dysfunction and increased mortality during polymicrobial sepsis

Abstract

Sepsis remains the leading cause of death in critically ill patients, despite modern advances in critical care. Intestinal barrier dysfunction may lead to secondary bacterial translocation and the development of the multiple organ dysfunction syndrome during sepsis. Cyclooxygenase (COX)-2 is highly upregulated in the intestine during sepsis, and we hypothesized that it may be critical in the maintenance of intestinal epithelial barrier function during peritonitis-induced polymicrobial sepsis. COX-2(-/-) and COX-2(+/+) BALB/c mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice chimeric for COX-2 were derived by bone marrow transplantation and underwent CLP. C2BBe1 cells, an intestinal epithelial cell line, were treated with the COX-2 inhibitor NS-398, PGD(2), or vehicle and stimulated with cytokines. COX-2(-/-) mice developed exaggerated bacteremia and increased mortality compared with COX-2(+/+) mice following CLP. Mice chimeric for COX-2 exhibited the recipient phenotype, suggesting that epithelial COX-2 expression in the ileum attenuates bacteremia following CLP. Absence of COX-2 significantly increased epithelial permeability of the ileum and reduced expression of the tight junction proteins zonula occludens-1, occludin, and claudin-1 in the ileum following CLP. Furthermore, PGD(2) attenuated cytokine-induced hyperpermeability and zonula occludens-1 downregulation in NS-398-treated C2BBe1 cells. Our findings reveal that absence of COX-2 is associated with enhanced intestinal epithelial permeability and leads to exaggerated bacterial translocation and increased mortality during peritonitis-induced sepsis. Taken together, our results suggest that epithelial expression of COX-2 in the ileum is a critical modulator of tight junction protein expression and intestinal barrier function during sepsis.

FIGURE 1

COX-2, but not COX-1, is upregulated in the ileum following peritonitis-induced polymicrobial sepsis. RNA was isolated from ileums of COX-2^+/+ mice following sham or CLP (n=2-3), reverse-transcribed, and qPCR performed for COX-2 (A) and COX-1 (B). Results were normalized to endogenous control 18S (p<0.05 by the Mann-Whitney test). (C) Protein was harvested from ileums of COX-2^+/+ mice (n=2-4) following sham or CLP and Western blot performed for COX-2 and COX-1. (D) Bar graphs represent protein levels (normalized for α-tubulin) as determined by densitometry. Protein levels are expressed as fold induction compared with COX-2^+/+ mice following sham (p<0.05 by the Kruskal-Wallis one-way ANOVA test followed by Dunn’s post test). (E) Representative COX-2 immunostaining of ileums harvested from COX-2^+/+ and COX-2^−/− mice 24 h following sham or CLP. Arrows represent positively stained inflammatory cells in the lamina propria and arrowheads represent positively stained epithelial cells in the ileum.

FIGURE 2

COX-2 deficient mice have increased mortality and exaggerated bacteremia following peritonitis-induced polymicrobial sepsis. (A) Male BALB/c COX-2^+/+ (■ n=10) and COX-2^−/− (□ n=9) underwent CLP with a 19-g needle (1 hole) in two independent experiments. Survival was monitored over 8 d. Data are expressed as percent of mice alive at each time point (p<0.05 by the log-rank test). (B) Blood was drawn from COX-2^+/+ (■ n=11) and COX-2^−/− (□ n=11) mice via right ventricular puncture and (C) MLN (COX-2^+/+ ■ n=7; COX-2^−/− □ n=12) and (D) organs (COX-2^+/+ ■ n=11; COX-2^−/− □ n=12) harvested 24 h following CLP. Blood and homogenized tissue were serially diluted and cultured on LB plates. Horizontal bars represent the median of each group and are from at least four independent experiments (p<0.05 by the Mann-Whitney test).

FIGURE 3

COX-2 deficient mice have increased ileal inflammation following peritonitis-induced polymicrobial sepsis. COX-2^+/+ and COX-2^−/− mice underwent CLP with a 19-g needle (1 hole). Ileums were harvested 48 h following CLP. (A) Representative H&E staining (a-d), Alcian blue staining (e-h), CD45 immunostaining (i-l), and Ly-6G immunostaining (m-p) in COX-2^+/+ and COX-2^−/− mice following sham surgery (left) and CLP (right). Arrows indicate cells staining positive for CD45 (l) and Ly-6G (p) in COX-2^−/− mice following CLP. (B) Histologic scoring of mucosal injury in H&E-stained ileums following sham (COX-2^−/− n=4; COX-2^+/+ n=5) and CLP (COX-2^−/− n=7; COX-2^+/+ n=9) from two independent experiments (*p<0.05 versus COX-2^−/− sham and **p<0.05 versus COX-2^+/+ sham by the χ² test for trend). (C) Quantitation of goblet cell number (3 mice per condition) per crypt-villus in COX-2^−/− and COX-2^+/+ mice following sham and CLP. Quantitation of CD45 (D) and Ly-6G (E) immunostaining in ileums of COX-2^−/− and COX-2^+/+ mice (2-5 mice per condition) demonstrates that COX-2^−/− mice have significantly increased neutrophilic inflammation in the ileum compared with COX-2^+/+ mice following CLP (*p<0.05 for COX-2^−/− mice after CLP versus COX-2^−/− mice after sham and versus COX-2^+/+ mice after CLP by the Mann-Whitney test).

FIGURE 4

COX-2 deficient mice have increased neutrophil infiltration of lamina propria of the ileum following peritonitis-induced polymicrobial sepsis. COX-2^+/+ and COX-2^−/− mice underwent CLP and ileums were harvested 24 h following sham (COX-2^−/− n=5; COX-2^+/+ n=6) and CLP (COX-2^−/− n=11; COX-2^+/+ n=11). (A) Representative flow cytometry plots from COX-2^+/+ and COX-2^−/− mice following sham and CLP. Neutrophils were quantified as Ly-6G⁺Ly-6C⁺ gated on CD45⁺CD11b⁺ cells. Flow cytometry of cells isolated from the lamina propria of the ileum demonstrates significantly increased numbers of neutrophils (Ly-6G⁺Ly-6C⁺) (B) and macrophages (F4/80⁺CD11b⁺) (C) in COX-2^−/− mice compared with COX-2^+/+ mice following CLP (p<0.05 by the Mann-Whitney test).

FIGURE 5

Absence of COX-2 in ileal parenchymal cells correlates with increased circulating bacterial counts and bacterial seeding of vital organs during polymicrobial sepsis. (A-B) Mice chimeric for COX-2 (COX-2^−/− → COX-2^+/+ (■ n=7), COX-2^+/+ → COX-2^−/− (□ n=7)) and (C-D) non-chimeric irradiated control mice (COX-2^+/+ → COX-2^+/+ (■ n=4), COX-2^−/− → COX-2^−/− (□ n=4)) were generated via BMT. Six weeks following BMT, mice underwent CLP with a 19-g needle (1 hole) in two independent experiments. Blood was drawn via right ventricular puncture and organs harvested 24 h following CLP. (A,C) Blood and (B,D) homogenized tissue were serially diluted and cultured on LB plates (p<0.05 by the Mann-Whitney test).

FIGURE 6

Deficiency of COX-2 increases intestinal epithelial permeability following peritonitis-induced polymicrobial sepsis. (A) COX-2^+/+ and COX-2^−/− mice underwent CLP with a 19-g needle (1 hole). Eighteen h following CLP (COX-2^+/+ n=7, COX-2^−/− n=8) and sham (COX-2^+/+ n=6, COX-2^−/− n=6), mice were gavaged with oral FD-4 and blood harvested after 6 h. Serum was isolated, FD-4 concentration measured, and normalized to sham FD-4 concentration. Data represent the median of three independent experiments (p<0.05 by the Mann-Whitney test). (B) COX-2^+/+ and COX-2^−/− mice underwent CLP with a 23-g needle (2 holes). Ileums were harvested 24 h following sham (COX-2^+/+ n=8, COX-2^−/− n=7) or CLP (COX-2^+/+ n=16, COX-2^−/− n=8) and permeability to FD-4 measured by the everted gut sac method. Data represent the median of at least three independent experiments (p<0.05 for COX-2^−/− mice vs. COX-2^+/+ mice following CLP, **p<0.05 for COX-2^−/− mice following CLP vs. sham, ‡p<0.05 for COX-2^+/+ mice following CLP vs. sham by the Mann-Whitney test).

FIGURE 7

Selective inhibition of COX-2 increases epithelial permeability in cytomix-stimulated intestinal epithelial cells. C2BBe1 cells were treated with vehicle, NS-398 (A), or SC-560 (C) and stimulated with CM in collagen-coated transwells. Epithelial permeability was assessed by measuring apical-to-basolateral clearance of FS and results normalized to vehicle. (B) TER was measured before and 48 h following CM stimulation. Data represent the median of at least two independent experiments performed in triplicate (p<0.05 by the Mann-Whitney test).

FIGURE 8

Absence of COX-2 leads to reduced expression of tight junction proteins in the ileum following CLP. (A-B) Ileums were harvested from COX-2^+/+ and COX-2^−/− mice 48 h following sham or CLP and total protein was isolated from ileal mucosa. Western blot analysis was performed for ZO-1, occludin, and claudin-1. Bar graphs represent protein levels (normalized for β-actin) as determined by densitometry and averaged for 2-4 mice per condition. Protein levels are expressed as the percentage of the levels in COX-2^+/+ mice following sham. Data are shown as the mean ± SD (p<0.05 as indicated and *p<0.05 for COX-2^+/+ mice after CLP vs. sham control, **p<0.05 for COX-2^−/− mice after CLP vs. sham control by unpaired t-test). (C) C2BBe1 cells were treated with NS-398 or vehicle and stimulated with CM in collagen-coated 8 well chamber slides. After 48 h, cells were fixed, permeabilized, and immunostained for ZO-1, occludin, and claudin-1. Confocal fluorescent microscopy was performed and photographs are representative of three independent experiments.

FIGURE 9

PGD₂ is upregulated in the ileum during peritonitis-induced polymicrobial sepsis. Ileums were harvested from COX-2^+/+ and COX-2^−/− mice 24 h following sham or CLP (n=4-8 per group), homogenized, and ELISA performed for PGD₂ in two independent experiments. PGD₂ is significantly upregulated in the ileum of COX-2^+/+ mice following CLP and COX-2^−/− mice have significantly reduced expression of PGD₂ in the ileum following CLP compared with COX-2^+/+ mice (p<0.05 by the Mann-Whitney test).

FIGURE 10

PGD₂ attenuates epithelial hyperpermeability and downregulation of ZO-1 expression induced by selective inhibition of COX-2. C2BBe1 cells were pre-treated with (A) PGD₂ or (D) BW245C (an agonist of the PGD₂ receptor DP1) prior to NS-398 or vehicle and stimulated with CM in collagen-coated transwells. Epithelial permeability was assessed by measuring apical-to-basolateral clearance of FS and results normalized to vehicle. (B) TER was measured before and 48 h following CM stimulation. Data represent the median of at least two independent experiments performed in triplicate (p<0.05 by the Mann-Whitney test). (C) Total protein was harvested from C2BBe1 cells 48 h following CM and Western blot analysis performed for ZO-1. Loading was quantified with an anti-α-tubulin Ab.