Trefoil factor 3 isolated from human breast milk downregulates cytokines (IL8 and IL6) and promotes human beta defensin (hBD2 and hBD4) expression in intestinal epithelial cells HT-29

Abstract

Trefoil factors (TFF) are secretory products of mucin producing cells. Th ey play a key role in the maintenance of the surface integrity of oral mucosa and enhance healing of the gastrointestinal mucosa by a process called restitution. TFF comprises the gastric peptides (TFF), spasmolytic peptide (TFF), and the intestinal trefoil factor (TFF). Th ey have an important and necessary role in epithelial restitution within the gastrointestinal tract. Signifi cant amounts of TFF are present in human milk. Th is study aimed to determine a possible correlation between TFF isolated from human breast milk and levels of cytokines (IL and IL) and defensins (hBD and hBD) in intestinal epithelial cells HT- treated with trefoil. Samples of human milk were collected within - weeks postpartum from healthy human mothers (--years-old) by manual breast massage, and TFF was purifi ed by ammonium sulfate precipitation, isoelectric precipitation, DEAE-chromatography, and gel fi ltration. In this work we measured the concentrations and mRNA levels of cytokines and defensins by immunoassay (ELISA) and semiquantitative RT-PCR technique, respectively. Also we measured the peroxidase activity. We present the fi rst evidence of human milk TFF purifi cation. Here we show that the presence of TFF isolated from milk strongly correlates with downregulation of IL and IL in human intestinal epithelial cells. On the other hand, TFF activated the epithelial cells in culture to produce beta defensins  (hBD) and beta defensins  (hBD). Th ese fi ndings suggest that TFF can activate intestinal epithelial cells and could actively participate in the immune system of breastfed babies by inducing the production of peptides related to innate defence, such as defensins.

Intestinal trefoil factor increased the Bcl-2 level in a necrotizingenterocolitis neonate rat model

“…Interestingly, ITF not only promoted cell proliferation and migration, but also special physical function; for example, it helped to stabilize the mucus layer by binding to mucous glycoprotein (14,15). Additionally, experiments have shown that ITF plays an important role in protecting the integrity of the intestinal mucosa and promoting its reconstruction after injury (16)(17)(18)(19). Mice lacking ITF did not heal well after mucosa injury.…”

Trefoil Factor Peptides and Gastrointestinal Function

“…In a rat necrotizing enterocolitis model, TFF3 improves inflammation by inhibiting the increase of Nf-κb, Il-1β, and Il-6, while enhancing the expression of Il-10, an anti-inflammatory cytokine (141). TFF3 inhibits IL-6 and IL-8 secretion induced by LPS and stimulates defensin production in the HT29 cell line in vitro (142). In addition to dampening pro-inflammatory cytokine amounts, TFF peptides themselves have been demonstrated to be regulated by cytokines or TLR signaling.…”

Trefoil factor 3 (TFF3) from human breast milk activates PAR-2 receptors, of the intestinal epithelial cells HT-29, regulating cytokines and defensins

“…Purifi ed TFF3 was subjected to electrophoresis on 15 % SDS-PAGE and immunoblotting as previously reported (3).…”

“…Membranes were washed 5 times with PBS-tween, blocked for 2 hours in 5 % skim milk in PBS-Tween, and incubated with 10 μg/ml of TFF3 (bait protein) in binding buffer (100 mMNaCl, 20 mMTris (pH 7.6), 0.5 mM EDTA, 10 % glycerol, 0.1 % Tween-20, 2 % skim milk powder) overnight at 4 °C. Bound TFF3 (bait protein) was detected by a primary antibody in a method similar to standard Immunoblotting described above (3). Membranes were stripped and were reprobed (the reprobed was done using standard immunobloting to determine the position of the PAR-2 (prey proteins) on the membrane).…”

“…Every one of them play a signifi cant role in the conservation and healing of human gastrointestinal tract. Previously we have reported that TFF3 isolated from breast milk produces down regulation of cytokines and defensins (3,4). This study aimed to determine the molecular mechanism involved in this process.…”