Potential immunoregulatory role of heme oxygenase-1 in human milk: a combined biochemical and molecular modeling approach

Human milk contains biological factors that are involved in a newborn's growthand immune system regulation. By integrating standard biochemical experimentalprotocols with computational methods, the present study investigates the presenceof heme oxygenase-1 (HO-1), a cytoprotective enzyme, in human milk at differentlevels of maturation and in milk formulae. Furthermore, we evaluated cytokine andglutathione S-transferase (GSH) levels. Samples were collected from colostrum (onDay 1 after birth), from transition milk (on Postdelivery Days 7 and 14) and frommature milk (on Day 30 after delivery) in 14 healthy women. HO-1 protein, GSH andcytokines levels were measured using enzyme-linked immunosorbent assay and flowcytometry. HO-1 protein levels were significantly higher in colostrum (1.33ng/ml; 5th centile 0.92; 95th centile 2.38) and in transition milk at 14 days(0.97 ng/ml; 5th centile 0.87; 95th centile 1.45) than in mature milk (0.9 ng/ml;5th centile 0.8; 95th centile 1.38). Levels of HO-1 in milk formulae were similarto those in colostrum. No significant differences in GSH content were observed inmature milk, transition milk and colostrum, whereas significantly higher GSHlevels were observed in milk formulae. No significant levels of cytokines, withthe exception of interleukin-8, were found. Computational studies on the possibleinteractions between HO-1 and CD91 were carried out by a battery of softwares,namely, GRAMM (version 1.03), DALI, CLUSTALW (version 2.0), PatchDock andFireDock, mutually counterchecking and validating each other. The computationalresults, the strong convergence (to the same "solution") of which finally leadsto an "experimental-like" character, showed that HO-1 may bind to CD91, thussuggesting its major role as a new chaperokine in immune response regulation.These findings, which connect and integrate biochemical data and computationaldata interpretation, represent a synergistic and powerful means of conductingbiological research