Cox-2 deletion in myeloid and endothelial cells, but not in epithelial cells, exacerbates murine colitis

Abstract

Patients with inflammatory bowel diseases are at increased risk for colorectal cancer. Pharmacological inhibition of cyclooxygenase (COX) function exacerbates symptoms in colitis patients. Animal models of colitis using Cox-2-knockout mice and COX inhibitors also indicate that COX-2 has a protective role against colon inflammation. However, because conventional Cox-2 deletion and COX-2 inhibitors eliminate COX-2 function in all cells, it has not been possible to analyze the role(s) of COX-2 in different cell types. Here, we use a Cox-2(flox) conditional knockout mouse to analyze the role of COX-2 expression in distinct cell types in the colon in response to dextran sulfate sodium (DSS)-induced colitis. We generated Cox-2 conditional knockouts in myeloid cells with LysMCre knock-in mice, in endothelial cells with VECadCreERT2 transgenic mice and in epithelial cells with VillinCre transgenic mice. When treated with DSS to induce colitis, both myeloid cell-specific and endothelial cell-specific Cox-2-knockout mice exhibited greater weight loss, increased clinical scores and decreased epithelial cell proliferation after DSS injury when compared with littermate controls. In contrast, epithelial-specific Cox-2 knockouts and control littermates did not differ in response to DSS. These results suggest that COX-2 expression in myeloid cells and endothelial cells, but not epithelial cells, is important for protection of epithelial cells in this murine colitis model.

Fig. 1.

Detection of Cox-2-expressing cells in colons of DSS-treated mice. (A and B) Ex vivo imaging of luciferase expression in the colon of a Cox-2^luc/+ mouse after 8 days of DSS treatment. Photo (A) shows a fixed, opened, methylene blue stained inflamed colon. Arrowheads indicate areas exhibiting ulceration. The color overlay on image (B) illustrates the photons per second emitted from the colon, as shown in the pseudocolor scale. (C–E) Immunofluorescent detection of COX-2-expressing cells. Dual-color immunofluorescent analyses of inflamed colons from DSS-treated wild-type mice were performed with anti-COX-2 antibody (red) and antibody to (C) F4/80 for macrophage (green), (D) CD34 for endothelial cells (green) and (E) pan-keratin for epithelial cells (green). Examples of double stained cells (yellow) are indicated by arrowheads.

Fig. 2.

Myeloid cell-specific Cox-2-knockout in Cox-2^flox/flox;LysM^Cre/+ mice. (A) Genomic DNA from peritoneal macrophages of Cox-2^flox/flox littermates (lane 1) and Cox-2^flox/flox;LysM^Cre/+ (lane 2) mice were analyzed by Southern blotting. The Cox-2^flox allele is 9.0 kb and the Cox-2^del allele is 8.0 kb. (B) COX-2 protein expression in LPS-treated peritoneal macrophages isolated from Cox-2^flox/flox littermate mice and Cox-2^flox/flox;LysM^Cre/+ mice. (C) PGE₂ production in the LPS-treated peritoneal macrophages shown in (B). (D) Absence of COX-2 expression in macrophages of DSS-treated Cox-2^flox/flox;LysM^Cre/+ mice. Colon sections from Cox-2^flox/flox;LysM^Cre/+ and littermate Cox-2^flox/flox mice treated with 2.5% DSS for 8 days were costained with anti-COX-2 (red) and anti-F4/80 (macrophage marker, green). Double-positive cells (arrowheads) are observed in inflamed colons of Cox-2^flox/flox mice but not in colons of Cox-2^flox/flox;LysM^Cre/+ mice. Examples of F4/80-positive and COX-2-negative cells are indicated by arrows.

Fig. 3.

DSS-induced colitis in myeloid cell-specific Cox-2-knockout mice. (A) Body weight change during 8 days of 2.5% DSS administration. Cox-2^flox/flox;LysM^Cre/+ mice (grey line, n = 7) loose significantly more weight than do littermate Cox-2^flox/flox mice (black line, n = 7, P < 0.01 by two-way analysis of variance analysis). Asterisks indicate the time points at which weights are significantly different by the Wilcoxon test. (B) Clinical scores during 8 days of 2.5% DSS administration. Cox-2^flox/flox;LysM^Cre/+ mice (grey line, n = 7) showed significantly higher clinical scores when compared with littermate Cox-2^flox/flox mice (black line, n = 7). P < 0.01 by two-way analysis of variance analysis. (C) Representative hematoxylin- and eosin-stained colon sections of each crypt damage grade, as described in Materials and Methods, following treatment for 8 days with DSS. The percentage distribution of damaged area in each grade is plotted for littermate Cox-2^flox/flox and Cox-2^flox/flox;LysM^Cre/+ mice. (D) Total crypt damage is calculated from each score and the distribution percentage. Averages ± SE are shown. Cox-2^flox/flox;LysM^Cre/+ mice show significantly higher scores by t-test (P < 0.01) compared with littermate Cox-2^flox/flox mice. (E) Representative images of EdU-labeled epithelial cells in colon sections from Cox-2^flox/flox;LysM^Cre/+ and littermate Cox-2^flox/flox mice treated for 5 days with 2.5% DSS. (F) Number of EdU-positive cells (10 fields per mouse) in colon sections. Averages ± SE are shown. The number of proliferating cells is significantly lower in colons sections from Cox-2^flox/flox;LysM^Cre/+ mice than in colon sections from littermate Cox-2^flox/flox mice (P < 0.01 by t-test). (G) Apoptotic epithelial cells in the colon crypts were determined by TUNEL assay. Representative images are shown for colon sections from mice treated for 3 days with 2.5% DSS. TUNEL-positive cells are observed only sparsely in the Cox-2^flox/flox colon (arrowhead). In contrast, clusters of apoptotic cells are detected in the Cox-2^flox/flox;LysM^Cre/+ colon (bracket). (H) Number of TUNEL-positive cells (10 fields per mouse) in colon sections. Averages ± SE are shown. The number of apoptotic cells is significantly greater in colon sections from Cox-2^flox/flox;LysM^Cre/+ mice than in colon sections from littermate Cox-2^flox/flox mice (P < 0.01 by t-test).

Fig. 4.

Endothelial cell-specific Cox-2-knockout in Cox-2^flox/flox;VECadCreERT2^Tg mice. (A) Polymerase chain reaction detection of Cox-2^flox recombination. Mice were injected with tamoxifen (five successive days) to activate CreERT2. Endothelial cells were enriched with anti-LSEC antibody-conjugated beads. Genomic DNAs were isolated from flow-through cells and the endothelial cell-enriched antibody-bead bound fractions and analyzed by polymerase chain reaction to detect Cox-2^flox (2067 bp) and Cox-2^del (651 bp) alleles. Lanes 1 and 2: Cox-2^flox/flox cells. Lanes 3 and 4: Cox-2^flox/flox;VECadCreERT2^Tg cells. Lanes 1 and 3: flow-through fraction; lanes 2 and 4: bound fraction. (B) Peritoneal macrophage genomic DNA analyzed by Southern blotting. Cox-2^flox (9.0 kb) and Cox-2^del (8.0 kb) alleles are present in Cox-2^flox/del heterozygous macrophages (lane 1). The Cox-2^del allele is absent in Cox-2^flox/flox;VECadCreERT2^Tg macrophages (lane 2). (C) Absence of COX-2 expression in endothelial cells of DSS-treated Cox-2^flox/flox;VECadCreERT2^Tg mice. Colon sections from Cox-2^flox/flox;VECadCreERT2^Tg and Cox-2^flox/flox littermates treated with 2.5% DSS for 8 days were costained with anti-COX-2 (red) and anti-CD34 (endothelial cell marker, green). Double-positive cells are observed on blood vessel structures in the colons of littermate Cox-2^flox/flox mice (arrowheads). In contrast, few—if any—CD34-positive cells express COX-2 in Cox-2^flox/flox;VECadCreERT2^Tg colons. (D) Colon sections similar to those in panel C were stained with anti-COX-2 (red) and anti-F4/80 (macrophage cell marker, green). Double-positive cells, in which membranes are positive for F4/80 and intracellular regions are positive for COX-2, are observed in sections from both Cox-2^flox/flox;VECadCreERT2^Tg and littermate Cox-2^flox/flox mice (arrowheads).

Fig. 5.

DSS-induced colitis in endothelial cell-specific Cox-2^flox/flox;VECadCreERT2^Tg-knockout mice. (A) Weight change during 2.5% DSS administration. Cox-2^flox/flox;VECadCreERT2^Tg mice (n = 10) loose significantly more weight than littermate Cox-2^flox/flox mice (n = 11, P < 0.01, two-way analysis of variance analysis). Asterisks indicate points with significantly different weights (Wilcoxon test, P < 0.01). (B) Clinical scores during 2.5% DSS administration. Cox-2^flox/flox;VECadCreERT2^Tg mice (n = 10) showed significantly higher scores compared with littermate Cox-2^flox/flox mice (n = 11, P < 0.01, two-way analysis of variance analysis). Asterisks and the double asterisk indicate times with significantly different scores (Wilcoxon test, P < 0.05 and P < 0.01, respectively). (C) Percentage distributions of damaged area of each grade, determined from hematoxylin- and eosin-stained sections, are plotted for Cox-2^flox/flox;VECadCreERT2^Tg and littermate Cox-2^flox/flox mice. (D) Total crypt damage scores. Averages ± SE are shown. Cox-2^flox/flox;VECadCreERT2^Tg mice show significantly higher scores (t-test, P < 0.01) compared with littermate Cox-2^flox/flox mice. (E) Representative images of BrdU-labeled epithelial cells in colon sections from mice treated for 5 days with 2.5% DSS. Clusters of BrdU-positive cells are observed at the bottom half of crypts in the colon of littermate Cox-2^flox/flox mice (asterisk). In contrast, positive cells are sparsely distributed in the colon of Cox-2^flox/flox;VECadCreERT2^Tg mice (arrowheads). (F) Number of BrdU-positive cells (10 fields per mouse) in colon sections. Averages ± SE are shown. The number of proliferating cells is significantly lower in colon sections from Cox-2^flox/flox;VECadCreERT2^Tg mice than in colon sections from littermate Cox-2^flox/flox mice (P < 0.01 by t-test). (G) Number of TUNEL-positive cells (10 fields per mouse) in colon sections of mice treated with 2.5% DSS for 3 days and for 5 days. Averages ± SE are shown. No statistically significant difference is observed between colon sections from Cox-2^flox/flox;VECadCreERT2^Tg mice and littermate Cox-2^flox/flox mice (P > 0.05 by t-test).

Fig. 6.

DSS-induced colitis in epithelial cell-specific Cox-2^flox/flox;VillinCre^Tg-knockout mice. (A) Genomic DNAs from colon epithelial cells of littermate Cox-2^flox/flox (lane 1) and Cox-2^flox/flox;VillinCre^Tg (lane 2) mice were analyzed by Southern blotting. (B) Weight change during 8 days of 2.5% DSS administration. No significant difference is observed between Cox-2^flox/flox;VillinCre^Tg mice (red line, n = 9) and littermate Cox-2^flox/flox mice (blue line, n = 10). (C) Clinical scores during 8 days of 2.5% DSS administration did not show any difference between Cox-2^flox/flox;VillinCre^Tg mice (red line, n = 9) and littermate Cox-2^flox/flox mice (blue line, n = 10).