Enhancement of intestinal inflammation in mice lacking interleukin 10 by deletion of the serotonin reuptake transporter

Abstract

Background: Enterochromaffin cells and enteric neurons synthesize and release serotonin (5-HT). Reuptake, mediated by a plasmalemmal transporter (SERT) terminates the action of released 5-HT. Serotonin secretion and serotonin reuptake transporter (SERT) expression have been reported to be decreased in TNBS-induced experimental colitis and in patients with ulcerative colitis. The present study was designed to utilize the transgenic deletion of SERT as a gain-of-function model to test the hypothesis that 5-HT is a pro-inflammatory mediator in experimental colitis.

Methods: Colitis was compared in animals with IL10(+/+)SERT(+/+) (wild-type), IL10(-/-)SERT(+/+), IL10(-/-)SERT(+/-), and IL10(-/-)/SERT(-/-) (double knockout) genotypes. Macroscopic and histological damage scores were evaluated after a time period of up to 15 weeks.

Key results: Serotonin reuptake transporter expression was significantly increased in the inflamed colons of IL-10(-/-) mice, which displayed intestinal damage and a minor decrement in general health. General health was significantly worse and intestinal inflammation was more severe in IL-10(-/-)SERT(+/-), and IL-10(-/-)SERT(-/-) mice than in IL-10(-/-)SERT(+/+) or wild-type animals. Regardless of the associated SERT genotype, the number of 5-HT-immunoreactive cells was decreased by approximately 55-65% in all mice lacking IL-10.

Conclusions & inferences: Our observations indicate that colitis associated with IL-10 deficient mice is enhanced when the IL-10 deficiency is combined with a SERT deficiency. The data support the concept that 5-HT is a pro-inflammatory mediator in the gut.

Figure 1

Expression of the serotonin reuptake transporter (SERT) in the colon of IL-10^−/− mice and controls. Real-time RT-PCR was used to quantify transcripts encoding SERT. Data are mean ± SEM (n = 3–4). ^aP < 0.05 compared with wild-type mice.

Figure 2

Global assessment of health and macroscopic inflammation of colon in wild-type, IL-10^−/−SERT^+/+, IL-10^−/−SERT^+/−, and IL-10^−/−SERT^−/− mice. (A) The global assessment score is increased in IL-10^−/−SERT^+/− and IL-10^−/−SERT^−/− mice when compared to wild-type mice (^aP < 0.05), but not when compared to IL-10^−/−SERT^+/+ mice. (B) Representative photomicrographs of abdomen of wild-type mice, IL-10^−/−SERT^+/+, IL-10^−/−SERT^+/−, and IL-10^−/−SERT^−/− mice are shown. Global assessment scores were obtained as described in Material and Methods. Data are mean ± SEM (n = 4).

Figure 3

Colonic inflammation in wild-type, IL-10^−/−SERT^+/+, IL-10^−/−SERT^+/−, and IL-10^−/−SERT^−/− mice. (A) Representative photomicrographs showing the histological appearance of the colon (H&E staining, 100× original magnification) in wild-type mice, IL-10^−/−SERT^+/+, IL-10^−/−SERT^+/−, and IL-10^−/−SERT^−/− mice. (B) Representative photomicrographs at higher magnification (400×) show infiltrating neutrophils and mononuclear cells in the submucosae of IL-10^−/−SERT^+/+, IL-10^−/−SERT^+/−, and IL-10^−/−SERT^−/− mice. (C) Inflammation scores obtained from the distal, middle, proximal colon. Data represent mean ± SEM (n = 4–6). ^aP < 0.05 compared to wild-type mice, ^bP < 0.05 compared to IL-10^−/−SERT^+/+ mice, ^cP < 0.05 compared to IL-10^−/−SERT^+/− mice. (D) Representative photomicrographs showing the simultaneous immunohistological identification of T and B lymphocytes infiltrating the mucosa. T lymphocytes were labeled with primary antibodies against CD3 (red) and B lymphocytes were labeled with antibodies against B220 (green). DNA was stained with DAPI to locate nuclei. Although lymphocytic infiltration of the mucosa was greater than normal (wild-type) in each of the strains of mice that lacked IL-10, lymphocytic infiltration of the mucosa was further potentiated when the IL-10 deficiency was combined with a partial (IL-10^−/−SERT^+/−) or complete knockout of serotonin reuptake transporter (SERT) (IL-10^−/−SERT^−/−).

Figure 4

Expression of transcripts encoding IL-6 and TNFα in the colons of wild-type, IL-10^−/−SERT^+/+, IL-10^−/−SERT^+/−, and IL-10^−/−SERT^−/− mice. (A) Abundance of transcripts encoding IL-6 is significantly greater in IL-10^−/−SERT^−/− than in wild-type mice (^aP < 0.01, ^bP < 0.01 compared to IL-10^−/−SERT^+/+ mice, ^cP < 0.01 compared to IL-10^−/−SERT^+/− mice). Data represent mean ± SEM (n = 4–6). (B) Abundance of transcripts encoding TNFα is significantly greater in IL-10^−/−SERT^−/− than in wild-type animals (^aP < 0.01). Data represent mean ± SEM (n = 4–6).

Figure 5

Serotonin immunoreactive cells in colon of wild-type, IL-10^−/−SERT^+/+, IL-10^−/−SERT^+/−, and IL-10^−/−SERT^−/− mice. Quantitative analysis of 5-HT immunoreactive cells in the proximal colon (400×). Data are shown as absolute numbers per field (mean ± SEM, n = 4–6). ^aP < 0.05 compared to wild-type mice.