Interferon-gamma inhibits intestinal restitution by preventing gap junction communication between enterocytes.
OBJECTIVE
Necrotizing enterocolitis (NEC) is characterized by interferon-gamma (IFN-gamma) release and inadequate intestinal restitution. Because enterocytes migrate together, mucosal healing may require interenterocyte communication via connexin 43-mediated gap junctions. We hypothesize that enterocyte migration requires interenterocyte communication, that IFN impairs migration by impairing connexin 43, and that impaired healing during NEC is associated with reduced gap junctions.


METHODS
NEC was induced in Swiss-Webster or IFN(-/-) mice, and restitution was determined in the presence of the gap junction inhibitor oleamide, or via time-lapse microscopy of IEC-6 cells. Connexin 43 expression, trafficking, and localization were detected in cultured or primary enterocytes or mouse or human intestine by confocal microscopy and (35)S-labeling, and gap junction communication was assessed using live microscopy with oleamide or connexin 43 siRNA.


RESULTS
Enterocytes expressed connexin 43 in vitro and in vivo, and exchanged fluorescent dye via gap junctions. Gap junction inhibition significantly reduced enterocyte migration in vitro and in vivo. NEC was associated with IFN release and loss of enterocyte connexin 43 expression. IFN inhibited enterocyte migration by reducing gap junction communication through the dephosphorylation and internalization of connexin 43. Gap junction inhibition significantly increased NEC severity, whereas reversal of the inhibitory effects of IFN on gap junction communication restored enterocyte migration after IFN exposure. Strikingly, IFN(-/-) mice were protected from the development of NEC, and showed restored connexin 43 expression and intestinal restitution.


CONCLUSIONS
IFN inhibits enterocyte migration by preventing interenterocyte gap junction communication. Connexin 43 loss may provide insights into the development of NEC, in which restitution is impaired.
Leaphart
Cynthia L
CL
Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
Qureshi
Faisal
F
Cetin
Selma
S
Li
Jun
J
Dubowski
Theresa
T
Baty
Catherine
C
Batey
Catherine
C
Beer-Stolz
Donna
D
Guo
Fengli
F
Murray
Sandra A
SA
Hackam
David J
DJ
D000818:Animals; D002450:Cell Communication / Q000187:drug effects*; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D018031:Connexin 43 / Q000378:metabolism; D020345:Enterocolitis, Necrotizing / Q000378:metabolism / Q000503:physiopathology* / Q000517:prevention & control; D020895:Enterocytes / Q000187:drug effects* / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects*; D006801:Humans; D007371:Interferon-gamma / Q000172:deficiency / Q000378:metabolism / Q000494:pharmacology*; D007422:Intestines / Q000187:drug effects / Q000503:physiopathology*; D051379:Mice; D018345:Mice, Knockout; D009829:Oleic Acids / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D014945:Wound Healing / Q000187:drug effects*
D018031:Connexin 43; D009829:Oleic Acids; C029407:oleylamide; D007371:Interferon-gamma