Lymphatic deletion of calcitonin receptor–like receptor exacerbates intestinal inflammation
Reema B. Davis, Daniel O. Kechele, Elizabeth S. Blakeney, John B. Pawlak, Kathleen M. Caron
Reema B. Davis, Daniel O. Kechele, Elizabeth S. Blakeney, John B. Pawlak, Kathleen M. Caron
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Research Article Gastroenterology

Lymphatic deletion of calcitonin receptor–like receptor exacerbates intestinal inflammation

Abstract

Lymphatics play a critical role in maintaining gastrointestinal homeostasis and in the absorption of dietary lipids, yet their roles in intestinal inflammation remain elusive. Given the increasing prevalence of inflammatory bowel disease, we investigated whether lymphatic vessels contribute to, or may be causative of, disease progression. We generated a mouse model with temporal and spatial deletion of the key lymphangiogenic receptor for the adrenomedullin peptide, calcitonin receptor–like receptor (Calcrl), and found that the loss of lymphatic Calcrl was sufficient to induce intestinal lymphangiectasia, characterized by dilated lacteals and protein-losing enteropathy. Upon indomethacin challenge, Calcrlfl/fl/Prox1-CreERT2 mice demonstrated persistent inflammation and failure to recover and thrive. The epithelium and crypts of Calcrlfl/fl/Prox1-CreERT2 mice exhibited exacerbated hallmarks of disease progression, and the lacteals demonstrated an inability to absorb lipids. Furthermore, we identified Calcrl/adrenomedullin signaling as an essential upstream regulator of the Notch pathway, previously shown to be critical for intestinal lacteal maintenance and junctional integrity. In conclusion, lymphatic insufficiency and lymphangiectasia caused by loss of lymphatic Calcrl exacerbates intestinal recovery following mucosal injury and underscores the importance of lymphatic function in promoting recovery from intestinal inflammation.

Authors

Reema B. Davis, Daniel O. Kechele, Elizabeth S. Blakeney, John B. Pawlak, Kathleen M. Caron

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Figure 3

Mice lacking lymphatic Calcrl do not recover from an acute mucosal injury challenge.

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Mice lacking lymphatic Calcrl do not recover from an acute mucosal injur...
(A) Representative images from whole small intestine (stomach and cecum attached) from tamoxifen-treated (TAM-treated) Calcrlfl/fl and Calcrlfl/fl/Prox1-CreERT2 mice 7 days after indomethacin (INDO) challenge. n = 7–8 animals in each group. Bottom panels show flushed intestines (stomach and cecum removed), and boxed areas are expanded as insets to show spot lesions. Scale bar: 1 cm. (B) Representative images of H&E-stained ilea of Calcrlfl/fl and Calcrlfl/fl/Prox1-CreERT2 mice after TAM treatment, 1 day after INDO challenge, and 7 days after INDO challenge. n = 5–10 animals in each group for each treatment. Scale bar: 50 μm. (C) Representative images of Masson’s trichrome stain showing fibrosis in ilea of TAM-treated Calcrlfl/fl and Calcrlfl/fl/Prox1-CreERT2 mice 7 days after INDO challenge. n = 7–8 animals in each group. Scale bar: 50 μm. (D) Relative expression of inflammatory markers in ilea of TAM-treated Calcrlfl/fl and Calcrlfl/fl/Prox1-CreERT2 mice 7 days after INDO challenge. n = 7–8 animals in each group. Quantitative data are represented as a box-and-whisker plot, with bounds from 25th to 75th percentile, median line, and whiskers ranging from minimum to maximum values of fold change over Calcrlfl/fl controls from 3 independent studies. Gapdh was used as housekeeping control. Significance was determined by 2-tailed, type 2 Student’s t test, *P < 0.05. (E) Quantification of small intestine length measured from proximal duodenum to distal ileum in Calcrlfl/fl and Calcrlfl/fl/Prox1-CreERT2 mice after TAM treatment, 1 day after INDO challenge, and 7 days after INDO challenge. n = 5–10 mice per group. Quantitative data are represented as a box-and-whisker plot, with bounds from 25th to 75th percentile, median line, and whiskers ranging from minimum to maximum values of gut length. Significance was determined by 2-tailed, type 2 Student’s t test, *P < 0.05. (F) Histopathological score of inflammation based on Table 1 (31) in Calcrlfl/fl and Calcrlfl/fl/Prox1-CreERT2 mice after TAM treatment, 1 day after INDO challenge, and 7 days after INDO challenge. n = 5–10 mice per group. Quantitative data are represented as a box-and-whisker plot, with bounds from 25th to 75th percentile, median line, and whiskers ranging from minimum to maximum inflammation scores. Significance was determined by 2-way ANOVA, *P < 0.05.