The PepT1-NOD2 signaling pathway aggravates induced colitis in mice

Abstract

Background & aims: The human di/tripeptide transporter human intestinal H-coupled oligonucleotide transporter (hPepT1) is abnormally expressed in colons of patients with inflammatory bowel disease, although its exact role in pathogenesis is unclear. We investigated the contribution of PepT1 to intestinal inflammation in mouse models of colitis and the involvement of the nucleotide-binding oligomerization domain 2 (NOD2) signaling pathway in the pathogenic activity of colonic epithelial hPepT1.

Methods: Transgenic mice were generated in which hPepT1 expression was regulated by the β-actin or villin promoters; colitis was induced using 2,4,6-trinitrobenzene sulfonic acid (TNBS) or dextran sodium sulfate (DSS) and the inflammatory responses were assessed. The effects of NOD2 deletion in the hPepT1 transgenic mice also was studied to determine the involvement of the PepT1-NOD2 signaling pathway.

Results: TNBS and DSS induced more severe levels of inflammation in β-actin-hPepT1 transgenic mice than wild-type littermates. Intestinal epithelial cell-specific hPepT1 overexpression in villin-hPepT1 transgenic mice increased the severity of inflammation induced by DSS, but not TNBS. Bone marrow transplantation studies showed that hPepT1 expression in intestinal epithelial cells and immune cells has an important role in the proinflammatory response. Antibiotics abolished the effect of hPepT1 overexpression on the inflammatory response in DSS-induced colitis in β-actin-hPepT1 and villin-hPepT1 transgenic mice, indicating that commensal bacteria are required to aggravate intestinal inflammation. Nod2-/-, β-actin-hPepT1 transgenic/Nod2-/-, and villin-hPepT1 transgenic/Nod2-/- littermates had similar levels of susceptibility to DSS-induced colitis, indicating that hPepT1 overexpression increased intestinal inflammation in a NOD2-dependent manner.

Conclusions: The PepT1-NOD2 signaling pathway is involved in aggravation of DSS-induced colitis in mice.

Figure 1. hPepT1 over-expression increases the susceptibility of mice to DSS-induced colitis

(A) Characterization of β-actin-hPepT1 mice. Western blotting analysis showing hPepT1 expression in tissues of WT and β-actin-hPepT1 animals (A-i). hPepT1 expression in colonic brush border membrane vesicles of WT and β-actin-hPepT1 animals (A-ii). Immunohistochemical analysis of hPepT1 using colonic sections from WT and β-actin-hPepT1 animals (A-iii). hPepT1-mediated uptake of KPV by colonic brush border membrane vesicles isolated from β-actin-hPepT1mice (A-iv). (B–F) Gender- and age-matched WT and β-actin-hPepT1 littermates were given water (H₂O) or 3% (w/v) DSS (DSS) for 8 days. Percentage change in body weight, clinical score, and colon length were assessed at day 8 post-treatment (B). Representative photographs obtained by endoscopy on the day of sacrifice (C). Histological score assessed at the end of DSS treatment. Representative H&E-stained distal colonic sections are shown. Bars =100 µm (D). Neutrophil infiltration into the colon was quantified by the measurement of MPO activity (E). Levels of colonic cytokine/chemokine mRNA measured by qRT-PCR (F). Data are means± SEMs (n=9/group/condition) from two experiments that yielded similar results (*P < .05; **P < .005).

Figure 2. hPepT1 over-expression increases the susceptibility of mice to TNBS-induced colitis

WT and β-actin-hPepT1 mice were given intrarectal water (vehicle) or TNBS dissolved in 50% (v/v) ethanol. Mice were sacrificed 2 days post-treatment. Percentage changes in body weight of β-actin-hPepT1 mice animals during treatment (*P < .05; **P < .005) vs. WT-TNBS animals. Clinical scores were assessed on the day of sacrifice (A). Representative photographs obtained by endoscopy on the day of sacrifice. Bars =100 µm (B). Representative H&E-stained distal colonic sections and histological scores assessed on the day of sacrifice (C). Levels of cytokine/chemokine mRNA as determined by qRT-PCR in the colons of control and TNBS-treated mice (D). Data are means ± SEMs (n=9/group/condition) from two experiments that yielded similar results (*P < .05; **P < .005; ***P < .001).

Figure 3. hPepT1 expression in IECs and in immune cells may play an important role in the pro-inflammatory response

Bone marrow transplants were performed (D: donor; R: recipient). RT-PCR (A) and Western blotting (B) analyses confirmed hPepT1 expression in bone marrow in mice receiving hPepT1 mouse-derived marrow. Mice were given intrarectal TNBS dissolved in 50% (v/v) ethanol, and were sacrificed 2 days post-treatment. Levels of colonic mRNAs encoding cytokines/chemokines were quantified by qRT-PCR in the colons of control and TNBS-treated mice (C). Data are means ± SEMs (n=9/group/condition) from two experiments that yielded similar results (*P < .05).

Figure 4. IEC-specific hPepT1 overexpression increases the susceptibility of mice to DSS-induced colitis

(A) Characterization of villin-hPepT1 mice. IEC-specific hPepT1 expression in villin-hPepT1 mice, but not in WT animals shown by western blotting (A-i). hPepT1 expression in colonic brush border membrane vesicles prepared from WT or villin-hPepT1 mice (A-ii). hPepT1 expression assessed by immunohistochemical analysis of colonic sections of WT and villin-hPepT1 animals (A-iii). hPepT1 mediated KPV transport in colonic brush border membrane vesicles prepared from villin-hPepT1 but not in WT mice (A-iv). (B–F) Gender- and age-matched WT and villin-hPepT1 littermates were given water (H₂O) or 3% (w/v) DSS for 8 days. Percent change in body weight, clinical score, and colon length were assessed at day 8 post-treatment (B). Representative photographs obtained by endoscopy on the day of sacrifice (C). Histological score assessed at the end of DSS treatment. Representative H&E-stained distal colonic sections are shown. Bars =100 µm (D). Neutrophil infiltration into the colon quantified by measurement of MPO activity (E). Levels of colonic mRNAs encoding cytokines/chemokines, as determined by qRT-PCR (F). Data are means ± SEMs (n=9/group/condition) from two experiments that yielded similar results (*P < .05; **P < .005; ***P < .001).

Figure 5. IEC-specific hPepT1 overexpression does not increase the susceptibility of mice to TNBS-induced colitis

WT and villin-hPepT1 mice were given intrarectal water (control) or TNBS dissolved in 50% (v/v) ethanol. Mice were sacrificed 2 days post-treatment. (A) Percent change in body weight during treatment. (B) Clinical score on the day of sacrifice. (C) Representative photographs obtained by endoscopy on the day of sacrifice. (D) Representative H&E-stained distal colonic sections and histological scores assessed on the day of sacrifice. (E) Levels of mRNAs encoding cytokines/chemokines (as determined by qRT-PCR) in the colons of control and TNBS-treated mice. Data are means ± SEMs (n=9/group/condition) from two experiments that yielded similar results. NS, not statistically significant.

Figure 6. hPepT1 overexpression that increases the susceptibility of mice to DSS-induced colitis requires the presence of bacterial flora

WT and β-actin hPepT1 mice were treated with broad-spectrum antibiotics A) Cultivable bacteria present in the feces before (pre-treatment) and after treatment (with antibiotics), and in water (vehicle)-treated mice, were counted. B) After antibiotic treatment, β-actin hPepT1 and WT mice were given water or a 3% (w/v) DSS solution for 8 days and the levels of colonic mRNAs encoding cytokines/chemokines were measured by qRT-PCR. Data are means ± SEMs (n=9/group/condition) from two experiments that yielded similar results (*P < .05; **P < .005; ***P < .001 vs. water). NS, not statically significant.

Figure 7. NOD2 expression increases the severity of colitis in hPepT1 transgenic mice

(A–C) Nod2^−/− and β-actin-hPepT1 Tg/Nod2^−/− mice were given water (control) or a 3% (w/v) DSS solution for 7 days. Percent change in body weight, assessed at day 7 post-treatment (A). Representative H&E-stained distal colonic sections are shown. Bars =100 µm (B). Levels of colonic mRNAs encoding cytokines/chemokines were analyzed by qRT-PCR (C). (D–F) Nod2^−/− and villin-hPepT1 Tg/Nod2^−/− mice were given water (control) or 3% (w/v) DSS solution for 7 days. Percentage change in body weight, assessed at day 7 post-treatment (D). Representative H&E-stained distal colonic sections are shown. Bars =100 mm (E). Levels of colonic mRNA encoding cytokines/chemokines were analyzed by qRT-PCR (F). Data are means ± SEMs (n=9/group/condition); the experiment was repeated twice with similar results (*P < .05; **P < .005; **).