Rotavirus double-stranded RNA induces apoptosis and diminishes wound repair in rat intestinal epithelial cells.
BACKGROUND
Recent studies have shown that toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA). Rotaviruses, having a dsRNA genome, infect intestinal epithelial cells (IEC) and cause acute gastroenteritis in young children. The aim of the present study was to clarify the pathophysiological function of rotavirus dsRNA in IEC.


METHODS
Expression of TLR3 mRNA or protein in IEC cell lines (IEC-6, HT-29, Caco-2) was assessed by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis or immunohistochemistry. Induction of cytokines (TNF-alpha, interferon-beta, interleukin-6) mRNA and activation of signal proteins (ERK1/2 MAPK and IkappaB-alpha) in IEC after stimulation with rotavirus dsRNA were assessed by RT-PCR or Western blot analysis. IEC-6 cells were wounded and cell migration into wound areas after stimulation with rotavirus dsRNA (1-25 microg/mL) was assessed. Induction of apoptosis after stimulation with rotavirus dsRNA was also assessed.


RESULTS
Expression of TLR3 mRNA and TLR3 protein was detected in IEC. Expression of TLR3 mRNA in IEC-6 tended to be up-regulated by exposure to IFN-gamma. Induction of cytokine mRNA and activation of the signal proteins were detected after stimulation with rotavirus dsRNA. Apoptosis was induced and epithelial migration into the wound area was dose-dependently diminished (44.1-94.4%, P < 0.01) by exposure to rotavirus dsRNA. Diminishment of wound repair was suppressed by anti-TLR3 antibody or caspase inhibitor.


CONCLUSIONS
Rotavirus dsRNA induces severe apoptosis and diminishes wound repair in IEC through TLR3, which might be involved in the pathogenesis of rotavirus-induced enteritis.
Sato
Akiko
A
Department of Internal Medicine, Akita University School of Medicine, Akita, Japan.
Iizuka
Masahiro
M
Nakagomi
Osamu
O
Suzuki
Manabu
M
Horie
Yasuo
Y
Konno
Shiho
S
Hirasawa
Fujiko
F
Sasaki
Kenji
K
Shindo
Kenichi
K
Watanabe
Sumio
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D016009:Chi-Square Distribution; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects*; D007150:Immunohistochemistry; D007371:Interferon-gamma / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000473:pathology; D012330:RNA, Double-Stranded / Q000502:physiology*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012401:Rotavirus; D018709:Statistics, Nonparametric; D051196:Toll-Like Receptor 3 / Q000502:physiology*; D015854:Up-Regulation; D014945:Wound Healing / Q000187:drug effects* / Q000502:physiology
D012330:RNA, Double-Stranded; D051196:Toll-Like Receptor 3; D007371:Interferon-gamma