Expression of corticotropin-releasing factor and urocortins in the normal and Schistosoma mansoni-infected mouse ileum

Abstract

Corticotropin-releasing factor (CRF) and urocortins (UCNs) are important ligands in the CRF signaling pathways, which are most known for their role in the hypothalamic-pituitary-adrenal stress axis. However, peripheral CRF signaling also has profound effects on gastrointestinal functions. Although the murine animal model is highly relevant for the exploration of this complexly balanced pathway via genetic manipulation, little is known about the expression of CRF and UCNs in the mouse intestine. This study aims to investigate the cellular localization of CRF and UCNs in the ileum and to explore whether and how this cellular expression is altered in conditions of intestinal Schistosoma mansoni-induced inflammation. The results show a distinct expression pattern for the different CRF receptor ligands in the ileum. CRF was located in nerve fibers and stromal cells. All UCNs were expressed in polymorphonuclear leukocytes. Furthermore, UCN2 and UCN3 were found in the musculature. During acute schistosomiasis, UCN1 showed an increased immunoreactivity in blood vessels and UCN3 was de novo expressed mainly in submucous neurons. Typical features of S. mansoni-inflamed ileum, such as nerve fiber sprouting, muscle layer thickening and granuloma formation thus all have an impact on the CRF signaling pathways. In conclusion, we outline for the first time the expression of CRF signaling ligands in the mouse ileum; our results point to important changes of this signaling system in S. mansoni-induced intestinal inflammation, which warrants further functional investigation with specific focus on CRF2, given the exclusive binding of UCN2 and UCN3 to this receptor.

Proof‐of‐concept: neonatal intravenous injection of adeno‐associated virus vectors results in successful transduction of myenteric and submucosal neurons in the mouse small and large intestine

“…Whole-mounts and cryosections were prepared as described before. 8,9 Tissue samples were preincubated in 0. , rabbit anti-S100B (1 : 5000; Dako, Glostrup, Denmark; Z0311), goat anti-calcitonin gene-related peptide (CGRP; 1 : 5000; Abcam ab36001), rabbit anti-glial fibrillary acidic protein (GFAP; 1 : 500; Dako Z0334), or rabbit anti-GFP (1 : 500; Abcam ab290). After rinsing with PBS, tissues were incubated with corresponding Cy3-conjugated secondary IgG antibodies raised in donkey (1 : 800; Jackson Immunoresearch, West Grove, PA, USA).…”

“…Antibodies were validated by appropriate negative control experiments as detailed previously. 10,11 In case of VIP and CGRP immunostaining for quantification, axonal transport was blocked with colchicine in organotypic culture enhancing the perikaryal localization of these neuropeptides. The culture medium was composed of 10% FCS, 100 U/mL penicillin-streptomycin, 50 lg/mL gentamycin, 2.5 lg/ mL amphotericin B, 1 lM nifedipine, 0.1 mg/mL colchicine in DMEM:F12 (Life Technologies).…”

“…Whole-mounts and cryosections were prepared as described before. 8,9 Tissue samples were preincubated in 0.1 M phosphatebuffered saline (PBS; pH 7.4) with 0.05% thimerosal, 10% normal horse serum, and 1% triton X-100. In case of anti-HuC/D staining this was supplemented with Mouse-On-Mouse blocking reagent (Vector Labs, Burlingame, CA, USA).…”

Gut-Brain Neuroendocrine Signaling Under Conditions of Stress—Focus on Food Intake-Regulatory Mediators

“…Injection of lipopolysaccharide, an immunological stressor, increases the expression of Ucn1, Ucn2 and Ucn3 mRNA in gastric mucosa and submucosa plus muscle layers (107) which is associated with the reduction of food intake under these conditions (53). Ucn1 and Ucn3 immunoreactivity in blood vessels and submucous neurons of the ileum is also increased following Schistosoma mansoni -induced inflammation (124). Likewise, blood monocyte-derived dendritic cells display largely increased Ucn1 mRNA and protein expression following stimulation with Bacteroides vulgatus or Fusobacterium varium (125).…”

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“…CRF is expressed in the colonic mucosal epithelia, lamina propria, crypts, and the enteric neurons (both bodies and fibers) in the rat colon [70], in healthy people and in patients with ulcerative colitis (UC) [71]. In the mice ileum, the expression pattern is slightly different with CRF primarily expressed in stromal cells and nerve fibers [72]. Specifically, enterochramffin cells (EC) that synthesize serotonin (5-HT) constitute a large proportion of CRF positive cells in the epithelia [70].…”