Modulation of toll-like receptor 7 and LL-37 expression in colon and breast epithelial cells by human beta-defensin-2.
Stroinigg. Nora N; Srivastava. Maya D MD
Key Findings
- In colon (CaCo‑2) cells, HBD‑2 lowered TLR‑7 and inflammatory markers while dramatically increasing LL‑37 protein levels.
- In breast (MCF‑7) cells, HBD‑2 raised both LL‑37 and TLR‑7 mRNA and protein, but did not affect many other defensins or TLRs.
- The response to HBD‑2 was cell‑type specific, indicating that the same peptide can have opposite immune effects in different tissues.
Practical Outcomes
- For self‑experimenters, the data suggest that supplementing with HBD‑2 (or related defensins) could modulate gut and breast tissue immunity, potentially increasing protective LL‑37 levels. However, the effects are tissue‑specific and the study used cell lines, so real‑world dosing and safety remain unclear. Until human trials are available, using HBD‑2 as a routine biohack for immunity is not yet justified.
Summary
The study shows that adding the antimicrobial protein HBD‑2 to colon and breast cells changes how those cells make other immune proteins, especially boosting the antimicrobial peptide LL‑37 and altering the activity of the immune sensor TLR‑7. These changes differ between gut and breast cells, suggesting HBD‑2 can reshape local immune responses.
Abstract
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.
Study Information
pubmed
2005