Figure 1.
CPE is expressed in EEC and affects colonic NPY and PYY levels.
(A–C) Immunofluorescence staining of colonic biopsies from wildtype mice for CPE and the EEC marker CgB (A), NPY (B) and PYY (C). (D–E) NPY (D) and PYY (E) neuropeptide level assayed by ELISA from LPS treated colon punch biopsies. n = 8 per genotype. (F–G) Determination of enteroendocrine cell numbers in the colonic (F) and ileal (G) mucosa by counting CgB, NPY and PYY positive epithelial cells per high power field (magnification 40x), 5 random HPF per animal, n = 6 per genotype. **p<0.01, ***p<0.001, ns = not significant, by t-test.
Figure 2.
CPE deficiency aggravates experimental chronic colitis.
(A) Calculation of the disease activity index (DAI) by determining clinical parameters of inflammation (body weight development, stool consistency, rectal bleeding) through 30 days of experimental colitis. n = 9 (cpe+/+), n = 8 (cpe−/−). (B-C) Determination of macroscopic colitis severity via mouse endoscopy. Representative endoluminal pictures of the distal colon on day 30 of experimental colitis (B) and calculation of the murine endoscopic index of colitis severity (MEICS) by analyzing mucosal morphology, stool consistency and shape of the vascular pattern via mouse endoscopy (C). n = 9 (cpe+/+), n = 8 (cpe−/−). (D–E) Determination of microscopic colitis severity via histology. Representative histological pictures of the distal colon on day 30 of experimental colitis (D) and calculation of the histology score by analyzing mucosal architecture and infiltration of immune cells (E). n = 8 per genotype. (F) Determination of expression level of TNF-α, IL-6 and KC in colonic punch biopsies by real time RT-PCR after 30 days of experimental colitis and at baseline. n = 8 per genotype. *p<0.05, **p<0.01, ***p<0.001 by t-test.
Figure 3.
Proinflammatory properties of colonic crypt supernatants of CPE-deficient mice.
(A) Experimental set-up for the acquirement and utilization of forskolin-stimulated supernatants of isolated colonic crypts. (B–C) Determination of KC (B) and IL-6 (C) transcript levels produced in MODE-K cells after incubation with LPS (50 ng/ml) and forskolin-stimulated supernatants of cpe+/+ and cpe−/− colonic crypts via RT-PCR. (D). KC transcript levels produced in MODE-K cells after incubation with forskolin-stimulated supernatants of of cpe+/+ and cpe−/− mice and LPS together with recombinant NPY +/− PYY (1 µM/ml). KC expression levels are expressed in percent of the Mean of cpe+/+. (E–F) BMDM migration via Boyden chamber assay. Representative pictures of migrated BMDM (E) and quantification (F) of BMDM migration towards supernatants of forskolin-stimulated colonic crypts of cpe+/+ and cpe−/− mice. *p<0.05, **p<0.01, ***p<0.001 by t-test.