Carboxypeptidase E Modulates Intestinal Immune Homeostasis and Protects against Experimental Colitis in Mice

<div><p>Enteroendocrine cells (EEC) produce neuropeptides, which are crucially involved in the maintenance of the intestinal barrier. Hence, EEC dysfunction is suggested to be involved in the complex pathophysiology of inflammatory bowel disease (IBD), which is characterized by decreased intestinal barrier function. However, the underlying mechanisms for EEC dysfunction are not clear and suitable models for a better understanding are lacking. Here, we demonstrate that Carboxypeptidase E (CPE) is specifically expressed in EEC of the murine colon and ileum and that its deficiency is associated with reduced intestinal levels of Neuropeptide Y (NPY) and Peptide YY (PYY), which are both produced by EEC. Moreover, <i>cpe^−/−</i> mice exhibit an aggravated course of DSS-induced chronic colitis compared to wildtype littermates. In addition, we observed elevated mucosal IL-6 and KC transcript levels already at baseline conditions in <i>cpe^−/−</i> mice. Moreover, supernatants obtained from isolated intestinal crypts of <i>cpe^−/−</i> mice lead to increased IL-6 and KC expression in MODE-K cells in the presence of LPS. This effect was reversible by co-administration of recombinant NPY, suggesting a CPE mediated immunosuppressive effect in the intestines by influencing the processing of specific neuropeptides. In this context, the chemotaxis of bone marrow derived macrophages towards respective supernatants was enhanced. In conclusion, our data point to an anti-inflammatory role of CPE in the intestine by influencing local cytokine levels and thus regulating the migration of myeloid immune cells into the mucosa. These findings highlight the importance of EEC for intestinal homeostasis and propose EEC as potential therapeutic targets in IBD.</p></div

CPE deficiency aggravates experimental chronic colitis.

<p>(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 (<i>cpe</i>^+/+), n = 8 (<i>cpe</i>^−/−). (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 (<i>cpe</i>^+/+), n = 8 (<i>cpe</i>^−/−). (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.</p

Proinflammatory properties of colonic crypt supernatants of CPE-deficient mice.

<p>(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 <i>cpe</i>^+/+ and <i>cpe</i>^−/− colonic crypts via RT-PCR. (D). KC transcript levels produced in MODE-K cells after incubation with forskolin-stimulated supernatants of of <i>cpe</i>^+/+ and <i>cpe</i>^−/− mice and LPS together with recombinant NPY +/− PYY (1 µM/ml). KC expression levels are expressed in percent of the Mean of <i>cpe^+/+</i>. (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 <i>cpe</i>^+/+ and <i>cpe</i>^−/− mice. *p<0.05, **p<0.01, ***p<0.001 by t-test.</p