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 6

Notch signaling is downregulated upon loss of lymphatic Calcrl.

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Notch signaling is downregulated upon loss of lymphatic Calcrl. (A) Rep...
(A) Representative images of delta-like canonical Notch ligand 4–stained (DLL4-stained) and lymphatic vessel endothelial hyaluronan receptor 1–stained (LYVE1-stained) ilea of tamoxifen-treated (TAM-treated) Calcrlfl/fl and Calcrlfl/fl/Prox1-CreERT2 mice 7 days after indomethacin (INDO) challenge. n = 8–10 animals in each group. Boxed regions highlight lacteals and submucosal lymphatic capillaries. Arrows point to DLL4 staining in lacteals. Arrowheads indicate DLL4 staining in crypts. Scale bar: 50 μm. (B and E) Relative expression of Notch pathway components in human neonatal dermal lymphatic endothelial cells after control siRNA or human calcitonin receptor–like receptor (CALCRL) siRNA treatment and vehicle (B) or 10 nM adrenomedullin (AM) treatment (E). Quantitative data are represented as box-and-whisker plots, with bounds from 25th to 75th percentile, median line, and whiskers ranging from minimum to maximum normalized fold change over Calcrlfl/fl from 4 independent studies. GAPDH, RN18S, and ACTB were used as housekeeping controls. Significance was determined by Student’s t test(tail = 2, type = 2), *P < 0.05. (C, D, F, and G) DLL4-stained (C and F) and activated NOTCH1-stained (D and G) human neonatal dermal lymphatic endothelial cells after control siRNA or CALCRL siRNA treated with vehicle (C and D) or 10 nM AM treatment (F and G). Images are representative of 3 independent knockdown experiments and treatment studies. Scale bar: 100 μm.