Korean J Urogenit Tract Infect Inflamm.  2014 Apr;9(1):27-33. 10.14777/kjutii.2014.9.1.27.

Modulation of Antimicrobial Peptide Human beta-defensin-3 by Toll-like Receptor Ligands in Vaginal Epithelial Cells

Affiliations
  • 1Department of Urology, Myongji Hospital, Goyang, Korea.
  • 2Functional Food Research Center, School of Life Sciences and Biotechnology, Korea University, Seoul, Korea.
  • 3Department of Urology, Chung-Ang University College of Medicine, Seoul, Korea. uromyung@cau.ac.kr
  • 4Department of Urology, Hallym University Dongtan Sacred Heart Hospital, Hallym University College of Medicine, Hwaseong, Korea.
  • 5Advanced Urogenital Disease Research Center, Chung-Ang University Hospital, Seoul, Korea.

Abstract

PURPOSE
Vaginal epithelial cells have always been exposed to various pathogens. However, this has not always caused clinical infection. In addition to a previously reported protection effect of the vagina, currently, the innate immune response is thought to be important as one of the causes explaining the phenomenon. Therefore, we investigated the innate immunity of the vagina and related mechanisms in infected vaginal epithelial cells focusing on the antimicrobial peptide human beta-defensin-3 (HBD-3).
MATERIALS AND METHODS
We investigated the signaling molecules, Toll-like receptors (TLRs), through which mammals sense infection in vaginal epithelial cells, with activation with lipopolysaccharide (LPS), Staphylococcus aureus peptidoglycan (PGN), or zymosan. Reverse transcriptase-polymerase chain reaction analysis of HBD-3 messenger RNA expression in vaginal epithelial cells after treatment with three pathogens was performed for investigation of pathogen-associated molecular patterns. Then, we also studied the following mechanism of innate immunity of the vagina focusing on HBD-3 in vaginal epithelial cells infected with gram-positive bacteria, gram-negative bacteria, or fungus.
RESULTS
Vaginal epithelial cells (VK2/E6E7 cells) constitutively expressed TLR2 and TLR4 and produced antimicrobial peptide HBD-3 upon activation with LPS, PGN, or zymosan. VK2/E6/E7 cells exposed to LPS, PGN, or zymosan showed increased p38 mitogen activated protein kinase (MAPK) activity. In addition, LPS-, PGN-, and zymosan-induced HBD-3 expression was attenuated by SB203580, a p38 MAPK inhibitor, emphasizing the importance of p38 MAPK in induction of HBD-3.
CONCLUSIONS
Vaginal epithelial cells may contribute to the host innate immune defense upon exposure to gram-negative bacteria, gram-positive bacteria, or fungi in the vagina by upregulation of HBD-3 expression.

Keyword

Vagina; Beta defensin 3, human; Toll-like receptors

MeSH Terms

Epithelial Cells*
Fungi
Gram-Negative Bacteria
Gram-Positive Bacteria
Humans
Immunity, Innate
Ligands*
Mammals
p38 Mitogen-Activated Protein Kinases
Peptidoglycan
Protein Kinases
RNA, Messenger
Staphylococcus aureus
Toll-Like Receptors*
Up-Regulation
Vagina
Zymosan
Ligands
Peptidoglycan
Protein Kinases
RNA, Messenger
Toll-Like Receptors
Zymosan
p38 Mitogen-Activated Protein Kinases

Figure

  • Fig. 1. Expression of Toll-like receptors (TLRs) and accessory molecules in cultured human vaginal epithelial cells. VK2/E6E7, a normal human vaginal epithelial cell line, was screened for mRNA expression of all known human TLRs and the accessory molecules myeloid differentiation protein 2 (MD-2), CD14, and myeloid differentiation primary response gene 88 (MyD88) by reverse transcriptase-polymerase chain reaction. The expression of TLR2 and TLR4 mRNA was detected in VK2/E6E7 cells. The expression of accessory molecules such as MyD88, CD14, and MD-2 was also detected. N.C: normal control, ACTB: β-actin.

  • Fig. 2. Pathogen-associated molecular patterns, representing gram-negative and gram-positive bacteria as well as fungal pathogens, induced human β-defensin-3 (HBD-3) in vaginal epithelial cells. Semiquantitative reverse transcriptase-polymerase chain reaction analysis of HBD-3 mRNA expression in immortalized vaginal epithelial cells after treatment with lipopolysaccharide (LPS), Staphylococcus aureus peptidoglycan (PGN), or zymosan was performed. LPS, PGN, and zymosan had an extremely strong stimulatory effect on HBD-3 mRNA expression in VK2/E6E7 cells at 3, 6, 12, and 24 hours after stimulation. ACTB: β-actin.

  • Fig. 3. The specific inhibitor of p38 MAPK (SB203580) attenuates lipopolysaccharide (LPS)-, Staphylococcus aureus peptidoglycan (PGN)-, and zymosan-induced human beta-defensin-3 (HBD-3) expression in VK2/E6E7 cells in the mRNA level (A) and the protein level (B).

  • Fig. 4. Lipopolysaccharide (LPS), Staphylococcus aureus peptidoglycan (PGN), and zymosan induced p38 mitogen-activated protein kinase (MAPK) phosphorylation in VK2/E6E7 cells. The activation of p38 MAPK by detecting the phosphor-p38 MAPK protein was observed.


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