ABSTRACT
BACKGROUND AND AIMS Methotrexate is an important tool in the arsenal of oncologists, gastroenterologists, and rheumatologists. At low doses it induces intestinal barrier dysfunction that may induce side effects such as gastrointestinal discomfort and liver injury. Previous studies suggest that lactoferrin can improve barrier function in a variety of contexts. This study set out to determine the mechanism of methotrexate induced barrier dysfunction and assess the effect of lactoferrin and other components of human breast milk on this dysfunction.
METHODS Using a murine enteroid model and Caco2 spheroids, we measured flux of basolateral-administered fluorescent dextran into the lumen. Barrier dysfunction was induced using methotrexate (220 nM) or lipopolysaccharide (20 nM). Human lactoferrin was added at 0.8 mg/ml (10 µM). RNAseq was performed on exposed samples.
RESULTS Lactoferrin blocks methotrexate-induced barrier dysfunction in murine enteroids. Similar results were observed when barrier dysfunction was induced in Caco2 spheroids with methotrexate and LPS, but not ML7. RNAseq revealed activation of TGF-β response genes and epithelial-mesenchymal transition (EMT) by methotrexate, which normalized in the presence of lactoferrin. TGF-β receptor inhibition (RepSox) blocked methotrexate induced barrier dysfunction in Caco2 spheroids. 20 nM TGF-β induced barrier dysfunction in Caco2 spheroids which was also inhibited by lactoferrin.
CONCLUSIONS Methotrexate induces barrier dysfunction by activation of an EMT program promoted by TGF-β signaling and inhibited by lactoferrin. Lactoferrin is also protective of barrier function in an LPS-induced model. The likely mechanism of this effect is blockade of EMT programs induced by TGF-β.
Footnotes
DISCLOSURES: None of the authors have financial disclosures.
GRANT SUPPORT: This research was supported by funds awarded to Z.J.G. by the Barbara Bakar Foundation, NIH (nos. U01CA199315 and DP2 HD080351-01), and the UCSF Center for Cellular Construction (no. DBI-1548297 an NSF Science and Technology Center). Z.J.G. is a Chan Zuckerberg BioHub Investigator. T.E.W. was supported by an NIH T32 Pediatric Training Grant. BD Aria III cell sorter is supported by National Cancer Institute Cancer Center Support Grant (P30CA082103) to the Laboratory for Cell Analysis, UCSF.
ABBREVIATIONS: Methotrexate (MTX), Lactoferrin (LTF), Epithelial to Mesenchymal Transition (EMT)
