Transforming growth factor-β2 is sequestered in preterm human milk by chondroitin sulfate proteoglycans

Namachivayam K1, Coffing HP2, Sankaranarayanan NV3, Jin Y3, MohanKumar K1, Frost BL4, Blanco CL5, Patel AL6, Meier PP7, Garzon SA8, Desai UR3,Maheshwari A9. Transforming growth factor-β2 is sequestered in preterm human milk by chondroitin sulfate proteoglycans. Am J Physiol Gastrointest Liver Physiol. 2015 Aug 1;309(3):G171-80. doi: 10.1152/ajpgi.00126.2015. PMID: 26045614 PMCID: PMC4525106

Human milk contains biologically important amounts of transforming growth factor-β2 isoform (TGF-β2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-β2 can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-β bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF-β2 (rTGF-β2) to milk prior to feeding. Milk-borne TGF-β bioactivity was measured by established luciferase reporter assays. Molecular interactions of TGF-β2 were investigated by nondenaturing gel electrophoresis and immunoblots, computational molecular modeling, and affinity capillary electrophoresis. Addition of rTGF-β2 (20-40 nM) to human preterm milk samples failed to increase TGF-β bioactivity in milk. Milk-borne TGF-β2 was bound to chondroitin sulfate (CS) containing proteoglycan(s) such as biglycan, which are expressed in high concentrations in milk. Chondroitinase treatment of milk increased the bioactivity of both endogenous and rTGF-β2, and consequently, enhanced the ability of preterm milk to suppress LPS-induced NF-κB activation in macrophages. These findings provide a mechanism for the normally low bioavailability of milk-borne TGF-β2 and identify chondroitinase digestion of milk as a potential therapeutic strategy to enhance the anti-inflammatory effects of preterm milk.

KEYWORDS: TGF-β2; breast milk; chondroitinase; inflammation; necrotizing enterocolitis