FOLH1/GCPII is elevated in IBD patients, and its inhibition ameliorates murine IBD abnormalities

Abstract

Recent gene-profiling analyses showed significant upregulation of the folate hydrolase (FOLH1) gene in the affected intestinal mucosa of patients with inflammatory bowel disease (IBD). The FOLH1 gene encodes a type II transmembrane glycoprotein termed glutamate carboxypeptidase II (GCPII). To establish that the previously reported increased gene expression was functional, we quantified the glutamate carboxypeptidase enzymatic activity in 31 surgical specimens and report a robust 2.8- to 41-fold increase in enzymatic activity in the affected intestinal mucosa of IBD patients compared with an uninvolved area in the same patients or intestinal mucosa from healthy controls. Using a human-to-mouse approach, we next showed a similar enzymatic increase in two well-validated IBD murine models and evaluated the therapeutic effect of the potent FOLH1/ GCPII inhibitor 2-phosphonomethyl pentanedioic acid (2-PMPA) (IC₅₀ = 300 pM). In the dextran sodium sulfate (DSS) colitis model, 2-PMPA inhibited the GCPII activity in the colonic mucosa by over 90% and substantially reduced the disease activity. The significance of the target was confirmed in FOLH1^-/- mice who exhibited resistance to DSS treatment. In the murine IL-10^-/- model of spontaneous colitis, daily 2-PMPA treatment also significantly reduced both macroscopic and microscopic disease severity. These results provide the first evidence of FOLH1/GCPII enzymatic inhibition as a therapeutic option for IBD.

Figure 1. Elevation of FOLH1/glutamate carboxypeptidase II activity in the diseased intestinal mucosa of patients with IBD.

FOLH1/glutamate carboxypeptidase II (FOLH1/GCPII) enzymatic activity was measured in involved (inflamed with active disease) and uninvolved (macroscopically normal) mucosal specimens from IBD patients or from non-IBD controls. (A) Significant enhancement of GCPII activity was observed in diseased mucosal specimens taken from Crohn’s disease (CD) and ulcerative colitis (UC) patients when compared with specimens from normal healthy controls in both colon and ileum. Data are shown as mean ± SEM (n = 31 samples) (*P < 0.05, **P < 0.01, 2-tailed t test). (B) Within the same IBD patient, a robust increase in enzymatic activity was observed in the diseased intestinal mucosa specimens when compared with that in an uninvolved region from the same patient. Data are presented as individual specimens (n = 19). The Arabic numbers refer to different patients. The pound sign indicates that an uninvolved normal region from this patient was not available.

Figure 2. Elevation of FOLH1/GCPII activity in murine models of colitis.

FOLH1/glutamate carboxypeptidase II (FOLH1/GCPII) enzymatic activity was evaluated in colon and ileum samples from two well-validated preclinical models of IBD. (A) Dextran sodium sulfate–treated (DSS-treated) mice showed significant enhancement of FOLH1/GCPII activity versus vehicle-treated mice in colon; a similar trend was observed in the ileum samples, although statistical significance was not achieved (*P < 0.05, 2-tailed t test). (B) IL-10^–/– mice showed a statistically significant enhancement of FOLH1/GCPII activity in both inflamed colon and ileum samples versus WT mice. Data are shown as mean ± SEM. Mouse samples (n = 4–7 for each group) were analyzed and assays were performed in triplicate (***P < 0.0001, 2-tailed t test).

Figure 3. 2-PMPA ameliorates dextran sodium sulfate–induced colitis.

(A) Daily 2-PMPA treatment (100 mg/kg i.p.) reduced colitis severity as indicated by the reduced disease activity index (DAI) scores which is a composite score of body weight, stool consistency, diarrhea and intestinal/rectal bleeding. Data are shown as mean ± SEM (n = 20 mice per group) (**P < 0.01, ***P < 0.001, 2-tailed t test). (B) 2-PMPA inhibits FOLH1/GCPII enzymatic activity in colonic mucosal extracts from dextran sodium sulfate–treated (DSS-treated) mice by > 90% confirming target engagement. Mouse samples (n = 4) were analyzed per group and assays were performed in triplicate. (***P < 0.001, 2-tailed t test). (C) Representative images of H&E-stained colon sections of 2-PMPA– and vehicle-treated mice with DSS-induced colitis (n = 5 per group). Untreated mice exhibited severe colitis, with disrupted epithelial linings, loss of crypts, thickening of the bowel wall, and massive infiltration of the inflammatory cells. 2-PMPA treatment led to protection of the crypts and drastic reduction of the inflammatory cell infiltration. Original magnification, ×100. M, mucosal layer; SM, submucosal; ML, muscular layer

Figure 4. FOLH1^–/– mice are protected against dextran sodium sulfate–induced colitis.

FOLH1^–/– and WT mice were given dextran sodium sulfate (DSS) in their drinking water for 7 days, and disease activity index (DAI) was monitored daily. Data are shown as mean ± SEM (n = 14 mice/group). (A) FOLH1^–/– mice showed significantly reduced DAI compared with WT mice (**P < 0.01, ***P < 0.001, 2-way ANOVA). (B) FOLH1^–/– mice showed significantly longer colons compared with WT, suggesting reduced inflammation (n = 5–14 mice per group) (***P < 0.001, 2-tailed t test). (C) Histological evaluation confirmed that FOLH1^–/– mice exhibited markedly reduced disease in response to DSS (n = 5 per group). WT mice exhibited thickening of the colon wall, including mucosa and muscular layers, as well as massive leukocyte infiltration, loss of crypts, and diminishing goblet cells, while the FOLH1^–/– mice showed relatively minor changes, with clearly defined crypts and visible goblet cells, as well as drastically reduced number of inflammatory cell infiltration. Original magnification, ×100. M, mucosal layer; SM, submucosal; ML, muscular layer.

Figure 5. 2-PMPA ameliorates disease activity in a IL-10^–/– model of spontaneous colitis.

IL-10^–/– mice (16–18 weeks old) were treated with daily 2-PMPA (100 mg/kg i.p.) for 3 weeks. Data are shown as mean ± SEM (n =18–20 mice per group). (A) 2-PMPA provided better stool consistency. (B) 2-PMPA improved colon weight (***P < 0.001, 2-tailed t test). (C) Histological evaluation showed that 2-PMPA treatment led to a healthier colon. Untreated mice exhibited marked thickening of the colon wall as a result of excessive hyperplasia, massive leukocyte infiltration, loss of crypts, and diminishing of goblet cells, while the 2-PMPA–treated group showed relatively minor changes. Original magnification, ×100. M, mucosal layer; SM, submucosal; ML, muscular layer.