Nucleotide-binding oligomerization domain-2 inhibits toll-like receptor-4 signaling in the intestinal epithelium.
OBJECTIVE
Factors that regulate enterocyte apoptosis in necrotizing enterocolitis (NEC) remain incompletely understood, although Toll-like receptor-4 (TLR4) signaling in enterocytes plays a major role. Nucleotide-binding oligomerization domain-2 (NOD2) is an immune receptor that regulates other branches of the immune system, although its effects on TLR4 in enterocytes and its role in NEC remain unknown. We now hypothesize that activation of NOD2 in the newborn intestine inhibits TLR4, and that failure of NOD2 signaling leads to NEC through increased TLR4-mediated enterocyte apoptosis.


METHODS
The effects of NOD2 on enterocyte TLR4 signaling and intestinal injury and repair were assessed in enterocytes lacking TLR4 or NOD2, in mice with intestinal-specific wild-type or dominant-negative TLR4 or NOD2, and in mice with NEC. A protein array was performed on NOD2-activated enterocytes to identify novel effector molecules involved.


RESULTS
TLR4 activation caused apoptosis in newborn but not adult small intestine or colon, and its intestinal expression was influenced by NOD2. NOD2 activation inhibited TLR4 in enterocytes, but not macrophages, and reversed the effects of TLR4 on intestinal mucosal injury and repair. Protection from TLR4-induced enterocyte apoptosis by NOD2 required a novel pathway linking NOD2 with the apoptosis mediator second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI (SMAC-DIABLO), both in vitro and in vivo. Strikingly, activation of NOD2 reduced SMAC-DIABLO expression, attenuated the extent of enterocyte apoptosis, and reduced the severity of NEC.


CONCLUSIONS
These findings reveal a novel inhibitory interaction between TLR4 and NOD2 signaling in enterocytes leading to the regulation of enterocyte apoptosis and suggest a therapeutic role for NOD2 in the protection of intestinal diseases such as NEC.
Richardson
Ward M
WM
Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Sodhi
Chhinder P
CP
Russo
Anthony
A
Siggers
Richard H
RH
Afrazi
Amin
A
Gribar
Steven C
SC
Neal
Matthew D
MD
Dai
Shipan
S
Prindle
Thomas
T
Branca
Maria
M
Ma
Congrong
C
Ozolek
John
J
Hackam
David J
DJ
D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000494:pharmacology; D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement; D004195:Disease Models, Animal; D019446:Endotoxemia / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020345:Enterocolitis, Necrotizing / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000517:prevention & control; D020895:Enterocytes / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D024101:Mitochondrial Proteins / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D053473:Nod2 Signaling Adaptor Protein / Q000819:agonists / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D040081:Protein Array Analysis; D051381:Rats; D012720:Severity of Illness Index; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C499605:Diablo protein, mouse; D024101:Mitochondrial Proteins; D016328:NF-kappa B; D053473:Nod2 Signaling Adaptor Protein; C495945:Nod2 protein, mouse; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D000119:Acetylmuramyl-Alanyl-Isoglutamine