The prostaglandin receptor EP4 suppresses colitis, mucosal damage and CD4 cell activation in the gut

Abstract

We used mice deficient in each of the eight types and subtypes of prostanoid receptors and examined the roles of prostanoids in dextran sodium sulfate-induced (DSS-induced) colitis. Among the prostanoid receptor-deficient mice, only EP4-deficient mice and not mice deficient in either DP, EP1, EP2, EP3, FP, IP, or TP developed severe colitis with 3% DSS treatment, which induced only marginal colitis in wild-type mice. This phenotype was mimicked in wild-type mice by administration of an EP4-selective antagonist (AE3-208). The EP4 deficiency impaired mucosal barrier function and induced epithelial loss, crypt damage, and aggregation of neutrophils and lymphocytes in the colon. Conversely, administration of an EP4-selective agonist (AE1-734) to wild-type mice ameliorated severe colitis normally induced with 7% DSS, while that of AE3-208 suppressed recovery from colitis and induced significant proliferation of CD4+ T cells. In vitro AE3-208 enhanced and AE1-734 suppressed the proliferation and Th1 cytokine production of lamina propria mononuclear cells from the colon. DNA microarray analysis revealed elevated expression of genes associated with immune response and reduced expression of genes with mucosal repair and remodeling in the colon of EP4-deficient mice. We conclude that EP4 maintains intestinal homeostasis by keeping mucosal integrity and downregulating immune response.

Figures

Figure 1

Induction of colitis by indomethacin in mice treated with 3% DSS and suppression of this effect by dm-PGE2. (a–c) Wild-type C57BL/6 mice were treated with either 3% DSS alone (diamonds), 3% DSS and indomethacin (squares), or 3% DSS, indomethacin, and dm-PGE2 (triangles), and monitored for body weight loss (a), diarrhea (b), and hemoccult (c) for 7 days. (d and e) On day 7, the mice were sacrificed. The colon was dissected for histological analysis with H&E staining (e), and the histological injury scores were obtained (d). Blood was collected for measurement of the WBC number (f) and HCT (g). The weights of spleens of the sacrificed animals are shown (h). N, mice treated with vehicle; D, mice treated with 3% DSS alone; D+I, mice treated with 3% DSS and indomethacin; D+I+E, mice treated with 3% DSS, indomethacin, and dm-PGE2. Data in a–d and f–h are means ± SEM from six animals. *P < 0.05 versus D (Dunnett). #P < 0.05 versus D+I (Dunnett multiple comparison test). Scale bars, 200 μm in e.

Figure 2

Induction of severe colitis by 3% DSS in EP4-deficient mice and not in mice deficient in other prostanoid receptors. (a–c) Wild-type C57BL/6 (filled circles), EP1–/– (filled triangles), EP2–/– (filled squares), EP3–/– (open circles), EP4+/+ (open triangles), and EP4–/– (open squares) mice were treated with 3% DSS for 7 days and monitored for body weight loss (a), diarrhea (b), and hemoccult (c). The numbers of mice used in this experiment were 25 (C57BL/6), 7 (EP1–/–), 10 (EP2–/–), 14 (EP3–/–), 16 (EP4–/–), and 12 (EP4+/+). (d–f) On day 7, EP4+/+ and EP4–/– mice treated with 3% DSS were sacrificed. The macroscopic examination of the large intestine is shown (d). The colon was dissected for histological analysis with H&E staining (e), and the histological injury scores were obtained (f). (g and h) Body weight loss (g) and clinical scores (h) of mice deficient in DP, FP, IP, and TP in 3% DSS colitis. DP–/–, FP+/+, FP–/–, IP–/–, and TP–/– mice were treated with 3% DSS for 7 days and monitored for body weight, diarrhea, and hemoccult. The body weight loss and clinical scores (diarrhea score plus hemoccult score) of these mice are compared with those of EP4+/+, EP4–/–, and C57BL/6 wild-type mice. The numbers of mice used in this experiment were 12 (EP4+/+), 16 (EP4–/–), 18 (C57BL/6), 11 (DP–/–), 5 (FP+/+), 4 (FP–/–), 8 (IP–/–), and 5 (TP–/–). Data in a–c and f–h are means ± SEM. *P < 0.05 versus EP4+/+ (t test) in a–c. Scale bars, 1 cm in d; 200 μm in upper and middle panel of e; 40 μm in lower panel of e.

Figure 3

Effect of EP4-selective drugs on DSS-induced colitis. (a) Structures of ONO-AE3-208 (left) and ONO-AE1-734 (right). (b and c) Reproduction of the EP4–/– phenotype in wild-type mice by administration of an EP4 antagonist. AE3-208, an EP4 antagonist, was added to 3% DSS in the drinking water and administered to C57BL/6 mice for 7 days. Body weight loss, diarrhea, and hemoccult scores on day 7 of 3% DSS-treated mice administered with vehicle (open bars) or AE3-208 (closed bars) is shown in b, and histological injury scores of the colon are shown in c. (d–f) Amelioration of high-dose (7%) DSS-induced colitis by an EP4 agonist. High-dose (7%) DSS was administered to C57BL/6 mice together with vehicle or AE1-734, an EP4 agonist, for 7 days. Body weight loss, diarrhea, and hemoccult scores on day 7 of 7% DSS-treated mice with vehicle (open bars) or the EP4 agonist (closed bars) are shown in d, and H&E staining of the colon (f) and the histological injury scores (e) are shown. Data are means ± SEM from five to ten mice. *P < 0.05 versus mice treated with 3% or 7% DSS alone (t test). Scale bars, 200 μm in f.

Figure 4

Impaired mucosal integrity caused by the EP4-deficiency. (a) Effect of the EP4 antagonist. C57BL/6 mice were treated with either vehicle (None), 3% DSS alone, AE3-208 (EP4 antagonist) alone, or 3% DSS and AE3-208 in combination for 1 day. FITC-dextran was administered by mouth, and the serum level was measured 4 hours later. Data are means ± SEM from six mice per group. *P < 0.05 versus the vehicle-treated mice (t test). (b) DSS-induced submucosal infiltration of FITC-dextran in EP4-deficient mice. FITC-dextran was administered by mouth to EP4+/+ and EP4–/– mice pretreated with 3% DSS for 1 day. Four hours later the colon was excised and subjected to fluorescent microscopic examination. Scale bars, 1 mm.

Figure 5

Impaired epithelial proliferation and enhanced CD4+ T cell proliferation in mice treated with an EP4 antagonist. Mice pretreated with 7% DSS for 7 days were then administered with either vehicle (a, d, e, and l) or the EP4 antagonist (AE3-208) (b, c, f–k, and m) for 3 days without DSS and sacrificed. BrdU was added intraperitoneally 2 hours before sacrifice. The colon was dissected for histological examination with H&E staining (a–c) and immunohistochemical analysis for BrdU (d–h), CD4 (i), CD8 (j), B220 (k), and IFN-γ (l and m). (e and g) Enlarged figures of d and f, respectively, showing the epithelial area; (h) an enlarged figure of f showing the submucosal area. (i–m) The submucosal area. Scale bars, 200 μm in a, b, d, f; 100 μm in c, e, g, h; 40 μm in i–m.

Figure 6

Modulation of the LPMNC proliferation and Th1 cytokine production by EP4 signaling. (a and b) LPMNCs were prepared from C57BL/6 mice (a) or from EP4–/– mice (b), cultured with either vehicle (None), the EP4 antagonist (AE3-208), the EP4 agonist (AE1-734), or indomethacin (IND) either alone or in indicated combination, and examined for the proliferation by measuring [3H] thymidine uptake. Data are means ± SEM (n = 3). *P < 0.05 versus None (Tukey multiple comparison test). #P < 0.05 versus IND (Tukey multiple comparison test). (c and d) LPMNCs were stimulated for 72 hours with vehicle, LPS, or LPS and ConA in combination in the presence of vehicle (None), the EP4 antagonist (AE3-208), or the EP4 agonist (AE1-734), and the levels of IFN-γ (c) and IL-2 (d) in the culture medium were measured with an ELISA kit. Data are means ± SEM (n = 3). *P < 0.05 versus None (Dunnett multiple comparison test).