Intestinal epithelial Toll-like receptor 4 regulates goblet cell development and is required for necrotizing enterocolitis in mice.
OBJECTIVE
Little is known about factors that regulate intestinal epithelial differentiation; microbial recognition receptors such as Toll-like receptor (TLR)4 might be involved. We investigated whether intestinal TLR4 regulates epithelial differentiation and is involved in development of necrotizing enterocolitis (NEC) of the immature intestine.


METHODS
Mice with conditional disruption of TLR4 in the intestinal epithelium and TLR4 knockout (TLR4(-/-)) mice were generated by breeding TLR4(loxp/loxp) mice with villin-cre and Ella-cre, respectively. Enterocytes that did not express or overexpressed TLR4 were created by lentiviral or adenoviral transduction. Intestinal organoids were cultured on tissue matrices. Bile acids were measured by colorimetric assays, and microbial composition was determined by 16S pyrosequencing. NEC was induced in 7- to 10-day-old mice by induction of hypoxia twice daily for 4 days.


RESULTS
TLR4(-/-) mice and mice with enterocyte-specific deletion of TLR4 were protected from NEC; epithelial differentiation into goblet cells was increased via suppressed Notch signaling in the small intestinal epithelium. TLR4 also regulates differentiation of goblet cells in intestinal organoid and enterocyte cell cultures; differentiation was increased on deletion of TLR4 and restored when TLR4 was expressed ectopically. TLR4 signaling via Notch was increased in intestinal tissue samples from patients with NEC, and numbers of goblet cells were reduced. 16S pyrosequencing revealed that wild-type and TLR4-deficient mice had similar microbial profiles; increased numbers of goblet cells were observed in mice given antibiotics. TLR4 deficiency reduced levels of luminal bile acids in vivo, and addition of bile acids to TLR4-deficient cell cultures prevented differentiation of goblet cells.


CONCLUSIONS
TLR4 signaling and Notch are increased in intestinal tissues of patients with NEC and required for induction of NEC in mice. TLR4 prevents goblet cell differentiation, independently of the microbiota. Bile acids might initiate goblet cell development.
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Chhinder P
CP
Sodhi
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Matthew D
MD
Neal
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Richard
R
Siggers
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Shonan
S
Sho
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Congrong
C
Ma
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Maria F
MF
Branca
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Thomas
T
Prindle
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Anthony M
AM
Russo
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Amin
A
Afrazi
Division of Newborn Medicine, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Misty
M
Good
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Rachel
R
Brower-Sinning
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Brian
B
Firek
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Michael J
MJ
Morowitz
Division of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
John A
JA
Ozolek
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
George K
GK
Gittes
Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Timothy R
TR
Billiar
Division of Pediatric Surgery, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Electronic address: david.hackam@chp.edu.
David J
DJ
Hackam
D000818:Animals; D000831:Animals, Newborn; D001647:Bile Acids and Salts / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D004195:Disease Models, Animal; D020345:Enterocolitis, Necrotizing / Q000209:etiology / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology / Q000517:prevention & control; D020397:Goblet Cells / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D006801:Humans; D000860:Hypoxia / Q000150:complications; D041943:Infant Formula; D007231:Infant, Newborn; D007421:Intestine, Small / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009940:Organoids; D034622:RNA Interference; D051381:Rats; D051880:Receptors, Notch / Q000378:metabolism; D015398:Signal Transduction; D046509:Tissue Culture Techniques; D051197:Toll-Like Receptor 4 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D001647:Bile Acids and Salts; D051880:Receptors, Notch; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4