Modulation of toll-like receptor 7 and LL-37 expression in colon and breast epithelial cells by human beta-defensin-2

Breast-feeding decreases maternal breast cancer risk. Breast-fed infants have fewer infections and inflammatory-allergic diseases. We recently found inducible antimicrobial and immunomodulatory protein human beta3-defensin 2 (HBD-2) in significant amounts in human milk. We investigated if HBD-2 could contribute to benefits of breast-feeding for the mother and the child by immunomodulating effects on breast and gut epithelial cells. Human CaCo-2 colon and MCF-7 breast cell lines were cultured for 16-48 hours in RPMI 1640 5% fetal calf serum with and without HBD-2 at 0.1, 0.5, and 1.0 microg/mL. RNA was extracted and reverse-transcription polymerase chain reaction (RT-PCR) and gel electrophoresis for toll-like receptor pathway members, antimicrobial peptides, and cytokines/receptors was performed. Primers were designed with www.ncbi.nlm.nih.gov and www.broad. mit.edu/cgibin/primer/primer3 www.cgi. Based on RT-PCR results, cells were stained by immunohistochemistry using anti-toll-like receptor (TLR)-7 and anti-LL37 antibodies and DAKO EnVision Plus kits. Supernatants were analyzed for interleukin (IL)-8 and liver and activation-regulated chemokine (LARC) using enzyme-linked immunosorbent assay. In CaCo-2, messenger RNA (mRNA) for TLR-7, IL-1R-associated kinase, alpha-defensins (human neutrophil peptides 1-3), and IL-8 were down-regulated; cathelicidin/LL37 and NFkappaBp65 were up-regulated. LARC mRNA and protein were detected after 48 hours. TLR-7 protein, LARC, and IL-8 decreased with HBD-2; LL-37 protein greatly increased. In MCF-7, mRNA for LL37, inhibitor of kappaBalpha, NFkappaBp65, Tollip, MyD88, IL-1R-associated kinase, and TLR-7 were up-regulated. LARC mRNA was turned off. TLR-7 protein was induced. LARC was not detected. IL-8 was barely detectable with or without HBD-2. beta-Defensins 1 and 2; alpha-defensins 5 and 6; TLRs 1, 2, 3, 4, 5, 6, 8, 9, and 10; nucleotide binding oligomerization domain protein-2, and CCR6 mRNA were unaffected. HBD-2 profoundly alters the innate immune response of breast and intestinal epithelial cells.