Ibuprofen slows migration and inhibits bowel colonization by enteric nervous system precursors in zebrafish, chick and mouse

Abstract

Hirschsprung Disease (HSCR) is a potentially deadly birth defect characterized by the absence of the enteric nervous system (ENS) in distal bowel. Although HSCR has clear genetic causes, no HSCR-associated mutation is 100% penetrant, suggesting gene-gene and gene-environment interactions determine HSCR occurrence. To test the hypothesis that certain medicines might alter HSCR risk we treated zebrafish with medications commonly used during early human pregnancy and discovered that ibuprofen caused HSCR-like absence of enteric neurons in distal bowel. Using fetal CF-1 mouse gut slice cultures, we found that ibuprofen treated enteric neural crest-derived cells (ENCDC) had reduced migration, fewer lamellipodia and lower levels of active RAC1/CDC42. Additionally, inhibiting ROCK, a RHOA effector and known RAC1 antagonist, reversed ibuprofen effects on migrating mouse ENCDC in culture. Ibuprofen also inhibited colonization of Ret+/− mouse bowel by ENCDC in vivo and dramatically reduced bowel colonization by chick ENCDC in culture. Interestingly, ibuprofen did not affect ENCDC migration until after at least three hours of exposure. Furthermore, mice deficient in Ptgs1 (COX 1) and Ptgs2 (COX 2) had normal bowel colonization by ENCDC and normal ENCDC migration in vitro suggesting COX-independent effects. Consistent with selective and strain specific effects on ENCDC, ibuprofen did not affect migration of gut mesenchymal cells, NIH3T3, or WT C57BL/6 ENCDC, and did not affect dorsal root ganglion cell precursor migration in zebrafish. Thus, ibuprofen inhibits ENCDC migration in vitro and bowel colonization by ENCDC in vivo in zebrafish, mouse and chick, but there are cell type and strain specific responses. These data raise concern that ibuprofen may increase Hirschsprung disease risk in some genetically susceptible children.

Maternal Risk Factors and Perinatal Characteristics for Hirschsprung Disease

“…9,11 In our study, we found that a parity of ≥3 children increases the risk of having a child with HSCR. On subanalysis for child sex, boys show the same pattern, but not girls, perhaps because of the small sample size and lack of power.…”

“…6 There have been speculations about the role of hypothyroidism, vitamin A deficiency, and maternal intake of Ibumetin or mycophenolate during pregnancy, but none of these associations have been confirmed. [7][8][9][10][11] The purpose of this study was to investigate the maternal risk factors and perinatal characteristics of HSCR in Sweden.…”

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies

“…using morpholinos and CRISPR knockdown technologies) of candidate genes implicated in ENS formation and for chemical screening of compounds that may affect enteric NCC migration, proliferation and/or differentiation in vivo . Although CRISPR technology is still in its infancy, particularly in the chick, chemical screening has recently been performed using zebrafish and chick to test the idea that certain medicines, taken during early human pregnancy, might alter HSCR risk (Schill et al, 2016). …”

Gene-environment interactions and the enteric nervous system: Neural plasticity and Hirschsprung disease prevention

“…Since only a single drug concentration was used for the zebrafish screen described above, it is reasonable to expect that other medicines would be found to affect ENCDC colonization of developing bowel if additional drug concentrations were tested (i.e., since the concentration tested may not be near the active concentration for many tested drugs). In support of this hypothesis, when drugs used by >0.5% of U.S. women during early pregnancy were tested at a range of concentrations on zebrafish, ibuprofen was found to slow ENCDC migration by altering actin cytoskeletal dynamics (Schill et al, 2015). Interestingly, inactivation of the cyclooxygenase enzymes that make prostaglandins (the primary therapeutic targets of ibuprofen) did not slow ENCDC colonization of fetal bowel in mice, suggesting that “off target” effects of ibuprofen lead to ENCDC bowel colonization defects.…”